Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention provides a non-naturally occurring microbial organism
having a 6-aminocaproic acid, caprolactam, hexametheylenediamine or
levulinic acid pathway. The microbial organism contains at least one
exogenous nucleic acid encoding an enzyme in the respective
6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic acid
pathway. The invention additionally provides a method for producing
6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic
acid. The method can include culturing a 6-aminocaproic acid, caprolactam
or hexametheylenediamine producing microbial organism, where the
microbial organism expresses at least one exogenous nucleic acid encoding
a 6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic
acid pathway enzyme in a sufficient amount to produce the respective
product, under conditions and for a sufficient period of time to produce
6-aminocaproic acid, caprolactam, hexametheylenediamine or levulinic
acid.

Claims:

1-282. (canceled)

283. Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic
acid is prepared from 2-oxoheptane-1,7-dioate (2-OHD), using at least one
biocatalyst.

284. Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic
acid is prepared from adipate semialdehyde, using at least one
biocatalyst.

285. Method according to claim 283, wherein the biocatalyst comprises an
enzyme capable of catalysing a transamination and/or a reductive
amination.

286. Method according to claim 285, wherein the enzyme capable of
catalysing a transamination and/or a reductive amination is selected from
the group of aminotransferases (E.C. 2.6.1) and amino acid dehydrogenases
(E.C. 1.4.1).

289. Method according to claim 286, wherein an aminotransferase is used
comprising an amino acid sequence according to: an enzyme from Vibrio
fluvialis, Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus
subtilis, or Pseudomonas aeruginosa that catalyses the conversion of
adipate semialdehyde to 6-aminocaproic acid; an enzyme from Vibrio
fluvialis, Pseudomonas aeruginosa, Pseudomonas syringae, Bacillus
subtilis, Rhodobaeter sphaeroides, Legionella pneumophila, Nitrosomonas
europaea, Neisseria gonorrhoeae, Pseudomonas aeruginosa, or
Rhodopseudomonas palustris that catalyses the conversion of
2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); a
gene product of gabT from Escherichia coli, puuE from Escherichia coli,
abat from Mus musculus, gabT from Pseudomonas fluorescens, or abat from
Sus scrofa; or a homologue of any of these sequences.

290. Method according to claim 283, wherein the biocatalyst comprises an
enzyme capable of catalysing the decarboxylation of an α-keto acid
or an amino acid.

291. Method according to claim 290, wherein the enzyme capable of
catalysing the decarboxylation is a decarboxylase (E.G. 4.1.1).

293. Method according to claim 290, wherein the enzyme capable of
catalysing the decarboxylation is enzyme from an organism or part thereof
selected from the group of Cucurbitaceae; Saccharomyces; Candida;
Hansenula; Kluyveromyces; Rhizopus; Neurospora; Zymomonas; Escherichia;
Mycobacterium; Clostridium; Lactobacillus; Streptococcus; Pseudomonas and
Lactococcus.

294. Method according to claim 290, wherein the enzyme capable of
catalysing the decarboxylation comprises an amino acid sequence according
to: an enzyme from Escherichia coli, Saccharomyces cerevisiae, Zymomonas
mobilis, Lactococcus lactis or Mycobacterium tuberculosis that catalyses
the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to adipate semialdehyde
or 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproic acid; a gene
product of pdc from Zymomonas mobilus, pdc1 from Saccharomyces
cerevisiae, pdc from Acetobacter pasteurians, pdc1 from Kluyveromyces
lactis, mdlC from Pseudomonas putida, mdlC from Pseudomonas aeruginosa,
dpgB from Pseudomonas stutzeri, ilvB-1 from Pseudomonas fluorescens, kgd
from Mycobacterium tuberculosis, kgd from Bradyrhizobium japonicum, kgd
from Mesorhizobium loti, kdcA from Lactococcus lactis, BCKDHB from Homo
sapiens, BCKDHA from Homo sapiens, BCKDHB from Bos taurus, BCKDHA from
Bos taurus, panD from Escherichia coli K12, panD from Corynebacterium
glutamicum or panD from Mycobacterium tuberculosis; or a homologue of any
of these sequences.

295. Method according to claim 290, wherein 2-OHD is biocatalytically
converted into adipate semialdehyde in the presence of a biocatalyst
capable of catalysing the decarboxylation of an α-keto acid, and
adipate semialdehyde is biocatalytically converted into 6-aminocaproic
acid in the presence of at least one amino donor and at least one
biocatalyst capable of catalysing a transamination and/or a reductive
amination of adipate semialdehyde.

296. Method according to claim 283, wherein 2-OHD is biocatalytically
converted into 2-aminoheptane-1,7-dioate (2-AHD) in the presence of at
least one amino donor and at least one biocatalyst capable of catalysing
a transamination and/or a reductive amination of 2-OHD thereby forming
2-AHD, and 2-AHD is biocatalytically converted into 6-aminocaproic acid
in the presence of a biocatalyst capable of catalysing the
decarboxylation of an amino acid.

297. Method according to claim 283, wherein the 2-OHD has been obtained
from a natural source.

304. A recombinant host cell according to claim 299, comprising one or
more nucleic acid sequences encoding one or more biocatalysts capable of
catalysing at least one reaction step in the preparation of
2-oxoheptane-1,7-dioate (2-OHD) from alpha-ketoglutarate.

305. A recombinant host cell according to claim 299, wherein the host
cell is selected from the group of Aspergillus, Penicillium,
Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus,
Corynebacterium, and Escherichia.

306. A micro-organism according to claim 299, comprising DNA containing a
nucleic acid sequence selected from the group of sequences represented by
any sequence selected from the group of: a gene from Vibrio fluvialis,
Bacillus weihenstephanensis, Pseudomonas aeruginosa, Bacillus subtilis,
or Pseudomonas aeruginosa that encodes an enzyme that catalyses the
conversion of adipate semialdehyde to 6-aminocaproic acid or a codon
optimized variant thereof; a gene from Vibrio fluvialis, Pseudomonas
aeruginosa, Pseudomonas syringae, Bacillus subtilis, Rhodobaeter
sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria
gonorrhoeae, Pseudomonas aeruginosa, or Rhodopseudomonas palustris that
encodes an enzyme that catalyses the conversion of
2-oxoheptane-1,7-dioate (2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD) or a
codon optimized variant thereof; a gabT gene from Escherichia coli, a
puuE gene from Escherichia coli, a abat gene from Mus musculus, a gabT
gene from Pseudomonas fluorescens, or a abat gene from Sus scrofa; a gene
from Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis,
Lactococcus lactis or Mycobacterium tuberculosis that encodes an enzyme
that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to
adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to
6-aminocaproic acid or a codon optimized variant thereof; a pdc gene from
Zymomonas mobilus, a pdc1 gene from Saccharomyces cerevisiae, a pdc gene
from Acetobacter pasteurians, a pdc1 gene from Kluyveromyces lactis, a
mdlC gene from Pseudomonas putida, a mdlC gene from Pseudomonas
aeruginosa, a dpgB gene from Pseudomonas stutzeri, an ilvB-1 gene from
Pseudomonas fluorescens, a kgd gene from Mycobacterium tuberculosis, a
kgd gene from Bradyrhizobium japonicum, a kgd gene from Mesorhizobium
loti, a kdcA gene from Lactococcus lactis, a BCKDHB gene from Homo
sapiens, a BCKDHA gene from Homo sapiens, a BCKDHB gene from Bos taurus,
a BCKDHA gene from Bos taurus, a panD gene from Escherichia coli K12, a
panD gene from Corynebacterium glutamicum or a panD gene from
Mycobacterium tuberculosis; and functional analogues thereof.

307. Polynucleotide comprising a nucleic acid sequence selected from the
group of sequences as identified in a codon optimized variant of a gene
from Vibrio fluvialis or Bacillus weihenstephanensis that encodes an
enzyme that catalyses the conversion of adipate semialdehyde to
6-aminocaproic acid; a codon optimized variant of a gene from Vibrio
fluvialis or Pseudomonas syringae that encodes an enzyme that catalyses
the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to
2-aminoheptane-1,7-dioate (2-AHD); a codon optimized variant of a gene
from Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis,
Lactococcus lactis or Mycobacterium tuberculosis that encodes an enzyme
that catalyses the conversion of 2-oxoheptane-1,7-dioate (2-OHD) to
adipate semialdehyde or 2-aminoheptane-1,7-dioate (2-AHD) to
6-aminocaproic acid; and functional analogues thereof.

[0002] The instant application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in its
entirety. Said ASCII copy, created on Jul. 11, 2012, is named
Sequence_Listing.txt and is 35,150 bytes in size.

BACKGROUND OF THE INVENTION

[0003] The present invention relates generally to biosynthetic processes,
and more specifically to organisms having adipate, hexamethylenediamine,
6-aminocaproic acid and caprolactam biosynthetic capability.

[0004] Adipic acid, a dicarboxylic acid, has a molecular weight of 146.14.
It can be used is to produce nylon 6,6, a linear polyamide made by
condensing adipic acid with hexamethylenediamine. This is employed for
manufacturing different kinds of fibers. Other uses of adipic acid
include its use in plasticizers, unsaturated polyesters, and polyester
polyols. Additional uses include for production of polyurethane,
lubricant components, and as a food ingredient as a flavorant and gelling
aid.

[0005] Historically, adipic acid was prepared from various fats using
oxidation. Some current processes for adipic acid synthesis rely on the
oxidation of KA oil, a mixture of cyclohexanone, the ketone or K
component, and cyclohexanol, the alcohol or A component, or of pure
cyclohexanol using an excess of strong nitric acid. There are several
variations of this theme which differ in the routes for production of KA
or cyclohexanol. For example, phenol is an alternative raw material in KA
oil production, and the process for the synthesis of adipic acid from
phenol has been described. The other versions of this process tend to use
oxidizing agents other than nitric acid, such as hydrogen peroxide, air
or oxygen.

[0006] In addition to hexamethylenediamine (HMDA) being used in the
production of nylon-6,6 as described above, it is also utilized to make
hexamethylene diisocyanate, a monomer feedstock used in the production of
polyurethane. The diamine also serves as a cross-linking agent in epoxy
resins. HMDA is presently produced by the hydrogenation of adiponitrile.

[0007] Caprolactam is an organic compound which is a lactam of
6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocaproic acid).
It can alternatively be considered cyclic amide of caproic acid. One use
of caprolactam is as a monomer in the production of nylon-6. Caprolactam
can be synthesized from cyclohexanone via an oximation process using
hydroxylammonium sulfate followed by catalytic rearrangement using the
Beckmann rearrangement process step.

[0008] Methods for effectively producing commercial quantities of
compounds such as hexamethylenediamine, 6-aminocaproic acid, levulinic
acid and carpolactamare described herein and include related advantages.

SUMMARY OF INVENTION

[0009] The invention provides a non-naturally occurring microbial organism
having a 6-aminocaproic acid, caprolactam or hexametheylenediamine
pathway. The microbial organism contains at least one exogenous nucleic
acid encoding an enzyme in the respective 6-aminocaproic acid,
caprolactam, hexametheylenediamine or levulinic acid pathway. The
invention additionally provides a method for producing 6-aminocaproic
acid, caprolactam or hexametheylenediamine. The method can include
culturing a 6-aminocaproic acid, caprolactam, hexametheylenediamine or
levulinic acid producing microbial organism, where the microbial organism
expresses at least one exogenous nucleic acid encoding a 6-aminocaproic
acid, caprolactam, hexametheylenediamine or levulinic acid pathway enzyme
in a sufficient amount to produce the respective product, under
conditions and for a sufficient period of time to produce 6-aminocaproic
acid, caprolactam, hexametheylenediamine or levulinic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows an exemplary pathway for adipate degradation in the
peroxisome of Penicillium chrysogenum.

[0011] FIG. 2 shows an exemplary pathway for adipate formation via a
reverse degradation pathway. Several options are provided for the final
conversion of adipyl-CoA to adipate.

[0023] FIG. 14 shows: A) the acetyl-CoA cycle of arginine biosynthesis.
Reactions (1) and (2) are catalyzed by ornithine acetyltransferase with
acetylglutamate synthase and ornithine acyltransferase functionality.
Reaction 3 is a lumped reaction catalyzed by acetylglutamate kinase,
N-acetylglutamylphosphate reductase, and acetylornithine
aminotransferase; B) the acetyl-CoA cycle of HMDA biosynthesis. Reactions
(1) and (2) are catalyzed by HMDA acetyltransferase. Reaction (3) is a
lumped reaction that includes all pathways to 6-acetamidohexanamine from
6-acetamidohexanoate shown in FIG. 13.

[0024] FIG. 15 shows the growth of E. coli in media containing various
concentrations of 6-ACA. E. coli was inoculated into media and grown in
either aerobic (left and right bars) or anaerobic (middle bars)
conditions. The cultures were grown for 48 hrs during the first trial and
30 hrs for a second trial under aerobic conditions (right bars).

[0025] FIG. 16 shows the tolerance of E. coli when exposed to 6-ACA.
Midlog (OD600=0.3, lower dashed line) or early stationary (OD600=0.6,
upper dashed line) cells were spun down and resuspended in fresh
M9-Glucose medium with various concentrations of 6-ACA. After overnight
growth, cultures were measured for growth by measuring OD600.

[0026] FIG. 17 shows the ethanol production from cultures exposed to
various concentrations of 6-ACA. Midlog or early stationary cells were
spun down and resuspended in fresh M9-Glucose medium with various
concentrations of 6-ACA. After overnight growth, cultures were measured
for growth by measuring OD600 and metabolic activity assayed by ethanol
production.

[0027] FIG. 18, panels A and B, show the growth in various concentrations
of 6-ACA with and without glycine betaine. Panel A. OD600 measurements of
medium inoculated with midlog cultures of E. coli with various
concentrations of 6-ACA with (right bars) and without (left bars) 2 mM
glycine betaine. Panel B. Photograph showing the growth of same cultures
in the anaerobic bottles.

[0028] FIG. 19 shows LC/MS analysis of in vitro thiolase reactions.
Succinyl-CoA and acetyl-CoA were added to His-tagged, purified thiolases
at a ratio of 2:1 (succinyl-CoA:acetyl-CoA). Reactions were analyzed by
LC/MS and quantified by comparison to a standard for acetoacetyl-CoA or
peak area determined for 3-oxoadipyl-CoA (β-ketoadipyl-CoA).

[0045] FIG. 36 shows the activity of CAR 889 and 891 using 20 mM Adipate.
Activity is shown as units per mg of total protein in the crude lysate.

[0046] FIG. 37 shows the activity of CAR 720, 889, 890, 891 using 50 mM
6-aminocaproate. Activity is shown as units per mg of total protein in
the crude lysate.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention is directed to the design and production of
cells and organisms having biosynthetic production capabilities for
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
The results described herein indicate that metabolic pathways can be
designed and recombinantly engineered to achieve the biosynthesis of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
in Escherichia coli and other cells or organisms. Biosynthetic production
of 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic
acid can be confirmed by construction of strains having the designed
metabolic genotype. These metabolically engineered cells or organisms
also can be subjected to adaptive evolution to further augment
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
biosynthesis, including under conditions approaching theoretical maximum
growth.

[0048] As disclosed herein, a number of metabolic pathways for the
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid are described. Two routes, the reverse adipate degradation
pathway and the 3-oxoadipate pathway, were found to be beneficial with
respect to: (i) the adipate yields (92% molar yield on glucose), (ii) the
lack of oxygen requirement for adipate synthesis, (iii) the associated
energetics, and (iv) the theoretical capability to produce adipate as the
sole fermentation product. Metabolic pathways for adipate production that
pass through a-ketoadipate or lysine are also described but are lower
yielding and require aeration for maximum production. A pathway for
producing either or both of 6-aminocaproate and caprolactam from
adipyl-CoA, a precursor in the reverse degradation pathway, is also
disclosed herein.

[0049] As disclosed herein, a number of exemplary pathways for
biosynthesis of adipate are described. One exemplary pathway involves
adipate synthesis via a route that relies on the reversibility of adipate
degradation as described in organisms such as P. chrysogenum (see
Examples I and II). A second exemplary pathway entails the formation of
3-oxoadipate followed by its reduction, dehydration and again reduction
to form adipate (see Examples III and IV). The adipate yield using either
of these two pathways is 0.92 moles per mole glucose consumed. The uptake
of oxygen is not required for attaining these theoretical maximum yields,
and the energetics under anaerobic conditions are favorable for growth
and product secretion. A method for producing adipate from
glucose-derived cis,cis-muconic acid was described previously (Frost et
al., U.S. Pat. No. 5,487,987, issued Jan. 30, 1996)(see Example V).
Advantages of the embodiments disclosed herein over this previously
described method are discussed. Metabolic pathways for adipate production
that pass through α-ketoadipate (Example VI) or lysine (Example
VII) precursors are lower yielding and require aeration for maximum
production. A pathway for producing either or both of 6-aminocaproate and
caprolactam from adipyl-CoA, a precursor in the reverse degradation
pathway, is described (see Example VIII and IX). Additional pathways for
producing adipate are described in Examples X and XI. Pathways for
producing any one, two, three or all four of 6-aminocaproate,
caprolactam, hexamethylenediamine and levulinic acid from succinyl-CoA
and acetyl-CoA are described in Examples XII, XXVIII. Several pathways
for the production of 6-aminocaproate from succinic semialdehyde and
pyruvate are described in Example XIX. Several pathways for the
production of hexamethylenediamine from 6-aminocaproate are described in
Examples XX and XXVII. A pathway for producing either or both
6-aminocaproate and hexamethylenediamine from glutamate is described in
Examples XXIV and XXV. Several pathways for the production of
hexamethylenediamine from glutaryl-CoA and at least one pathway for
production of 6-aminocaproate from glutaryl-CoA are described in Examples
XXIV and XXV. A pathway for producing 6-aminocaproate from homolysine is
described in Example XXV. Pathways for producing hexamethylenediamine
from 2-amino-7-oxosubarate are described in Example XXIV. Several
pathways for producing 6-aminocaproate are described in Example XXV.
Exemplary genes and enzymes required for constructing microbes with these
capabilities are described as well as methods for cloning and
transformation, monitoring product formation, and using the engineered
microorganisms for production.

[0050] As disclosed herein, six different pathways for adipic acid
synthesis using glucose/sucrose as a carbon substrate are described. For
all maximum yield calculations, the missing reactions in a given pathway
were added to the E. coli stoichiometric network in SimPheny that is
similar to the one described previously (Reed et al., Genome Biol. 4:R54
(2003)). Adipate is a charged molecule under physiological conditions and
was assumed to require energy in the form of a proton-based symport
system to be secreted out of the network. Such a transport system is
thermodynamically feasible if the fermentations are carried out at
neutral or near-neutral pH. Low pH adipic acid formation would require an
ATP-dependant export mechanism, for example, the ABC system as opposed to
proton symport. The reactions in the pathways and methods of
implementation of these pathways are described in Examples I-XI.

[0051] As used herein, the term "non-naturally occurring" when used in
reference to a microbial organism or microorganism of the invention is
intended to mean that the microbial organism has at least one genetic
alteration not normally found in a naturally occurring strain of the
referenced species, including wild-type strains of the referenced
species. Genetic alterations include, for example, modifications
introducing expressible nucleic acids encoding metabolic polypeptides,
other nucleic acid additions, nucleic acid deletions and/or other
functional disruption of the microbial genetic material. Such
modifications include, for example, coding regions and functional
fragments thereof, for heterologous, homologous or both heterologous and
homologous polypeptides for the referenced species. Additional
modifications include, for example, non-coding regulatory regions in
which the modifications alter expression of a gene or operon. Exemplary
metabolic polypeptides include enzymes within a 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid biosynthetic pathway.

[0053] As used herein, the term "isolated" when used in reference to a
microbial organism is intended to mean an organism that is substantially
free of at least one component as the referenced microbial organism is
found in nature. The term includes a microbial organism that is removed
from some or all components as it is found in its natural environment.
The term also includes a microbial organism that is removed from some or
all components as the microbial organism is found in non-naturally
occurring environments. Therefore, an isolated microbial organism is
partly or completely separated from other substances as it is found in
nature or as it is grown, stored or subsisted in non-naturally occurring
environments. Specific examples of isolated microbial organisms include
partially pure microbes, substantially pure microbes and microbes
cultured in a medium that is non-naturally occurring.

[0054] As used herein, the terms "microbial," "microbial organism" or
"microorganism" is intended to mean any organism that exists as a
microscopic cell that is included within the domains of archaea, bacteria
or eukarya. Therefore, the term is intended to encompass prokaryotic or
eukaryotic cells or organisms having a microscopic size and includes
bacteria, archaea and eubacteria of all species as well as eukaryotic
microorganisms such as yeast and fungi. The term also includes cell
cultures of any species that can be cultured for the production of a
biochemical.

[0055] As used herein, the term "CoA" or "coenzyme A" is intended to mean
an organic cofactor or prosthetic group (nonprotein portion of an enzyme)
whose presence is required for the activity of many enzymes (the
apoenzyme) to form an active enzyme system. Coenzyme A functions in
certain condensing enzymes, acts in acetyl or other acyl group transfer
and in fatty acid synthesis and oxidation, pyruvate oxidation and in
other acetylation.

[0056] As used herein, "adipate," having the chemical formula
--OOC--(CH2)4-COO-- (see FIG. 2) (IUPAC name hexanedioate), is the
ionized form of adipic acid (IUPAC name hexanedioic acid), and it is
understood that adipate and adipic acid can be used interchangeably
throughout to refer to the compound in any of its neutral or ionized
forms, including any salt forms thereof. It is understood by those
skilled understand that the specific form will depend on the pH.

[0057] As used herein, "6-aminocaproate," having the chemical formula
--OOC--(CH2)5-NH2 (see FIGS. 8 and 12), is the ionized form of
6-aminocaproic acid (IUPAC name 6-aminohexanoic acid), and it is
understood that 6-aminocaproate and 6-aminocaproic acid can be used
interchangeably throughout to refer to the compound in any of its neutral
or ionized forms, including any salt forms thereof. It is understood by
those skilled understand that the specific form will depend on the pH.

[0058] As used herein, "caprolactam" (IUPAC name azepan-2-one) is a lactam
of 6-aminohexanoic acid (see FIG. 8).

[0059] As used herein, "hexamethylenediamine," also referred to as
1,6-diaminohexane or 1,6-hexanediamine, has the chemical formula
H2N(CH2)6NH2 (see FIGS. 10, 11 and 13).

[0060] As used herein, the term "substantially anaerobic" when used in
reference to a culture or growth condition is intended to mean that the
amount of oxygen is less than about 10% of saturation for dissolved
oxygen in liquid media. The term also is intended to include sealed
chambers of liquid or solid medium maintained with an atmosphere of less
than about 1% oxygen.

[0061] As used herein, the term "osmoprotectant" when used in reference to
a culture or growth condition is intended to mean a compound that acts as
an osmolyte and helps a microbial organism as described herein survive
osmotic stress. Osmoprotectants include, for example, betaines, amino
acids, and the sugar trehalose. Non-limiting examples of such are glycine
betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-carnitine and ectoine.

[0062] As used herein, the term "growth-coupled" when used in reference to
the production of a biochemical is intended to mean that the biosynthesis
of the referenced biochemical is produced during the growth phase of a
microorganism. In a particular embodiment, the growth-coupled production
can be obligatory, meaning that the biosynthesis of the referenced
biochemical is an obligatory product produced during the growth phase of
a microorganism.

[0063] As used herein, "metabolic modification" is intended to refer to a
biochemical reaction that is altered from its naturally occurring state.
Metabolic modifications can include, for example, elimination of a
biochemical reaction activity by functional disruptions of one or more
genes encoding an enzyme participating in the reaction. Sets of exemplary
metabolic modifications are described herein (see Example XXX).

[0064] As used herein, the term "gene disruption," or grammatical
equivalents thereof, is intended to mean a genetic alteration that
renders the encoded gene product inactive. The genetic alteration can be,
for example, deletion of the entire gene, deletion of a regulatory
sequence required for transcription or translation, deletion of a portion
of the gene which results in a truncated gene product, or by any of
various mutation strategies that inactivate the encoded gene product. One
particularly useful method of gene disruption is complete gene deletion
because it reduces or eliminates the occurrence of genetic reversions in
the non-naturally occurring microorganisms of the invention.

[0065] "Exogenous" as it is used herein is intended to mean that the
referenced molecule or the referenced activity is introduced into the
host microbial organism. The molecule can be introduced, for example, by
introduction of an encoding nucleic acid into the host genetic material
such as by integration into a host chromosome or as non-chromosomal
genetic material such as a plasmid. Therefore, the term as it is used in
reference to expression of an encoding nucleic acid refers to
introduction of the encoding nucleic acid in an expressible form into the
microbial organism. When used in reference to a biosynthetic activity,
the term refers to an activity that is introduced into the host reference
organism. The source can be, for example, a homologous or heterologous
encoding nucleic acid that expresses the referenced activity following
introduction into the host microbial organism. Therefore, the term
"endogenous" refers to a referenced molecule or activity that is present
in the host. Similarly, the term when used in reference to expression of
an encoding nucleic acid refers to expression of an encoding nucleic acid
contained within the microbial organism. The term "heterologous" refers
to a molecule or activity derived from a source other than the referenced
species whereas "homologous" refers to a molecule or activity derived
from the host microbial organism. Accordingly, exogenous expression of an
encoding nucleic acid of the invention can utilize either or both a
heterologous or homologous encoding nucleic acid.

[0066] It is understood that when more than one exogenous nucleic acid is
included in a microbial organism that the more than one exogenous nucleic
acids refers to the referenced encoding nucleic acid or biosynthetic
activity, as discussed above. It is further understood, as disclosed
herein, that such more than one exogenous nucleic acids can be introduced
into the host microbial organism on separate nucleic acid molecules, on
polycistronic nucleic acid molecules, or a combination thereof, and still
be considered as more than one exogenous nucleic acid. For example, as
disclosed herein a microbial organism can be engineered to express two or
more exogenous nucleic acids encoding a desired pathway enzyme or
protein. In the case where two exogenous nucleic acids encoding a desired
activity are introduced into a host microbial organism, it is understood
that the two exogenous nucleic acids can be introduced as a single
nucleic acid, for example, on a single plasmid, on separate plasmids, can
be integrated into the host chromosome at a single site or multiple
sites, and still be considered as two exogenous nucleic acids. Similarly,
it is understood that more than two exogenous nucleic acids can be
introduced into a host organism in any desired combination, for example,
on a single plasmid, on separate plasmids, can be integrated into the
host chromosome at a single site or multiple sites, and still be
considered as two or more exogenous nucleic acids, for example three
exogenous nucleic acids. Thus, the number of referenced exogenous nucleic
acids or biosynthetic activities refers to the number of encoding nucleic
acids or the number of biosynthetic activities, not the number of
separate nucleic acids introduced into the host organism.

[0067] The non-naturally occurring microbial organisms of the invention
can contain stable genetic alterations, which refers to microorganisms
that can be cultured for greater than five generations without loss of
the alteration. Generally, stable genetic alterations include
modifications that persist greater than 10 generations, particularly
stable modifications will persist more than about 25 generations, and
more particularly, stable genetic modifications will be greater than 50
generations, including indefinitely.

[0068] In the case of gene disruptions, a particularly useful stable
genetic alteration is a gene deletion. The use of a gene deletion to
introduce a stable genetic alteration is particularly useful to reduce
the likelihood of a reversion to a phenotype prior to the genetic
alteration. For example, stable growth-coupled production of a
biochemical can be achieved, for example, by deletion of a gene encoding
an enzyme catalyzing one or more reactions within a set of metabolic
modifications. The stability of growth-coupled production of a
biochemical can be further enhanced through multiple deletions,
significantly reducing the likelihood of multiple compensatory reversions
occurring for each disrupted activity.

[0069] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein, are
described with reference to a suitable host organism such as E. coli and
their corresponding metabolic reactions or a suitable source organism for
desired genetic material such as genes for a desired metabolic pathway.
However, given the complete genome sequencing of a wide variety of
organisms and the high level of skill in the area of genomics, those
skilled in the art will readily be able to apply the teachings and
guidance provided herein to essentially all other organisms. For example,
the E. coli metabolic alterations exemplified herein can readily be
applied to other species by incorporating the same or analogous encoding
nucleic acid from species other than the referenced species. Such genetic
alterations include, for example, genetic alterations of species
homologs, in general, and in particular, orthologs, paralogs or
nonorthologous gene displacements.

[0070] An ortholog is a gene or genes that are related by vertical descent
and are responsible for substantially the same or identical functions in
different organisms. For example, mouse epoxide hydrolase and human
epoxide hydrolase can be considered orthologs for the biological function
of hydrolysis of epoxides. Genes are related by vertical descent when,
for example, they share sequence similarity of sufficient amount to
indicate they are homologous, or related by evolution from a common
ancestor. Genes can also be considered orthologs if they share
three-dimensional structure but not necessarily sequence similarity, of a
sufficient amount to indicate that they have evolved from a common
ancestor to the extent that the primary sequence similarity is not
identifiable. Genes that are orthologous can encode proteins with
sequence similarity of about 25% to 100% amino acid sequence identity.
Genes encoding proteins sharing an amino acid similarity less that 25%
can also be considered to have arisen by vertical descent if their
three-dimensional structure also shows similarities. Members of the
serine protease family of enzymes, including tissue plasminogen activator
and elastase, are considered to have arisen by vertical descent from a
common ancestor.

[0071] Orthologs include genes or their encoded gene products that
through, for example, evolution, have diverged in structure or overall
activity. For example, where one species encodes a gene product
exhibiting two functions and where such functions have been separated
into distinct genes in a second species, the three genes and their
corresponding products are considered to be orthologs. For the production
of a biochemical product, those skilled in the art will understand that
the orthologous gene harboring the metabolic activity to be introduced or
disrupted is to be chosen for construction of the non-naturally occurring
microorganism. An example of orthologs exhibiting separable activities is
where distinct activities have been separated into distinct gene products
between two or more species or within a single species. A specific
example is the separation of elastase proteolysis and plasminogen
proteolysis, two types of serine protease activity, into distinct
molecules as plasminogen activator and elastase. A second example is the
separation of mycoplasma 5'-3' exonuclease and Drosophila DNA polymerase
III activity. The DNA polymerase from the first species can be considered
an ortholog to either or both of the exonuclease or the polymerase from
the second species and vice versa.

[0072] In contrast, paralogs are homologs related by, for example,
duplication followed by evolutionary divergence and have similar or
common, but not identical functions. Paralogs can originate or derive
from, for example, the same species or from a different species. For
example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble
epoxide hydrolase (epoxide hydrolase II) can be considered paralogs
because they represent two distinct enzymes, co-evolved from a common
ancestor, that catalyze distinct reactions and have distinct functions in
the same species. Paralogs are proteins from the same species with
significant sequence similarity to each other suggesting that they are
homologous, or related through co-evolution from a common ancestor.
Groups of paralogous protein families include HipA homologs, luciferase
genes, peptidases, and others.

[0073] A nonorthologous gene displacement is a nonorthologous gene from
one species that can substitute for a referenced gene function in a
different species. Substitution includes, for example, being able to
perform substantially the same or a similar function in the species of
origin compared to the referenced function in the different species.
Although generally, a nonorthologous gene displacement will be
identifiable as structurally related to a known gene encoding the
referenced function, less structurally related but functionally similar
genes and their corresponding gene products nevertheless will still fall
within the meaning of the term as it is used herein. Functional
similarity requires, for example, at least some structural similarity in
the active site or binding region of a nonorthologous gene product
compared to a gene encoding the function sought to be substituted.
Therefore, a nonorthologous gene includes, for example, a paralog or an
unrelated gene.

[0074] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic
capability, those skilled in the art will understand with applying the
teaching and guidance provided herein to a particular species that the
identification of metabolic modifications can include identification and
inclusion or inactivation of orthologs. To the extent that paralogs
and/or nonorthologous gene displacements are present in the referenced
microorganism that encode an enzyme catalyzing a similar or substantially
similar metabolic reaction, those skilled in the art also can utilize
these evolutionally related genes. In gene disruption strategies,
evolutionally related genes can also be disrupted or deleted in a host
microbial organism, paralogs or orthologs, to reduce or eliminate
activities to ensure that any functional redundancy in enzymatic
activities targeted for disruption do not short circuit the designed
metabolic modifications.

[0075] Orthologs, paralogs and nonorthologous gene displacements can be
determined by methods well known to those skilled in the art. For
example, inspection of nucleic acid or amino acid sequences for two
polypeptides will reveal sequence identity and similarities between the
compared sequences. Based on such similarities, one skilled in the art
can determine if the similarity is sufficiently high to indicate the
proteins are related through evolution from a common ancestor. Algorithms
well known to those skilled in the art, such as Align, BLAST, Clustal W
and others compare and determine a raw sequence similarity or identity,
and also determine the presence or significance of gaps in the sequence
which can be assigned a weight or score. Such algorithms also are known
in the art and are similarly applicable for determining nucleotide
sequence similarity or identity. Parameters for sufficient similarity to
determine relatedness are computed based on well known methods for
calculating statistical similarity, or the chance of finding a similar
match in a random polypeptide, and the significance of the match
determined. A computer comparison of two or more sequences can, if
desired, also be optimized visually by those skilled in the art. Related
gene products or proteins can be expected to have a high similarity, for
example, 25% to 100% sequence identity. Proteins that are unrelated can
have an identity which is essentially the same as would be expected to
occur by chance, if a database of sufficient size is scanned (about 5%).
Sequences between 5% and 24% may or may not represent sufficient homology
to conclude that the compared sequences are related. Additional
statistical analysis to determine the significance of such matches given
the size of the data set can be carried out to determine the relevance of
these sequences.

[0076] Exemplary parameters for determining relatedness of two or more
sequences using the BLAST algorithm, for example, can be as set forth
below. Briefly, amino acid sequence alignments can be performed using
BLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters: Matrix:
0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0;
wordsize: 3; filter: on. Nucleic acid sequence alignments can be
performed using BLASTN version 2.0.6 (Sep. 16, 1998) and the following
parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2;
x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in
the art will know what modifications can be made to the above parameters
to either increase or decrease the stringency of the comparison, for
example, and determine the relatedness of two or more sequences.

[0077] Disclosed herein are non-naturally occurring microbial organisms
capable of producing adipate, 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid. For example, an adipate pathway
can be a reverse adipate degradation pathway (see Examples I and II). For
example, a non-naturally occurring microbial organism can have an adipate
pathway including at least one exogenous nucleic acid encoding an adipate
pathway enzyme expressed in a sufficient amount to produce adipate, the
adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase,
3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,
5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or
phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase
or adipyl-CoA hydrolase. In addition, an adipate pathway can be through a
3-oxoadipate pathway (see Examples III and IV). A non-naturally occurring
microbial organism can have an adipate pathway including at least one
exogenous nucleic acid encoding an adipate pathway enzyme expressed in a
sufficient amount to produce adipate, the adipate pathway including
succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase,
3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoate
reductase.

[0078] Additionally, a non-naturally occurring microbial organism can have
a 6-aminocaproic acid pathway including at least one exogenous nucleic
acid encoding a 6-aminocaproic acid pathway enzyme expressed in a
sufficient amount to produce 6-aminocaproic acid, the 6-aminocaproic acid
pathway including CoA-dependent aldehyde dehydrogenase and transaminase
(see Examples VIII and IX). Alternatively, 6-aminocaproate dehydrogenase
can be used to convert adipate semialdehyde to form 6-aminocaproate (see
FIG. 8). A non-naturally occurring microbial organism can also have a
caprolactam pathway including at least one exogenous nucleic acid
encoding a caprolactam pathway enzyme expressed in a sufficient amount to
produce caprolactam, the caprolactam pathway including CoA-dependent
aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase,
and amidohydrolase (see Examples VIII and IX).

[0079] As disclosed herein, a 6-aminocaproic acid or caprolactam producing
microbial organism can produce 6-aminocaproic acid and/or caprolactam
from an adipyl-CoA precursor (see FIG. 8 and Examples VIII and IX).
Therefore, it is understood that a 6-aminocaproic acid or caprolactam
producing microbial organism can further include a pathway to produce
adipyl-CoA. For example an adipyl-CoA pathway can include the enzymes of
FIG. 2 that utilize succinyl-CoA and acetyl-CoA as precursors through the
production of adipyl-CoA, that is, lacking an enzyme for the final step
of converting adipyl-CoA to adipate. Thus, one exemplary adipyl-CoA
pathway can include succinyl-CoA:acetyl-CoA acyl transferase,
3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and
5-carboxy-2-pentenoyl-CoA reductase.

[0080] In addition, as shown in FIG. 1, an adipate degradation pathway
includes the step of converting adipate to adipyl-CoA by an adipate CoA
ligase. Therefore, an adipyl-CoA pathway can be an adipate pathway that
further includes an enzyme activity that converts adipate to adipyl-CoA,
including, for example, adipate-CoA ligase activity as in the first step
of FIG. 1 or any of the enzymes in the final step of FIG. 2 carried out
in the reverse direction, for example, any of adipyl-CoA synthetase (also
referred to as adipate Co-A ligase), phosphotransadipylase/adipate
kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. An
enzyme having adipate to adipyl-CoA activity can be an endogenous
activity or can be provided as an exogenous nucleic acid encoding the
enzyme, as disclosed herein. Thus, it is understood that any adipate
pathway can be utilized with an adipate to adipyl-CoA enzymatic activity
to generate an adipyl-CoA pathway. Such a pathway can be included in a
6-aminocaproic acid or caprolactam producing microbial organism to
provide an adipyl-CoA precursor for 6-aminocaproic acid and/or
caprolactam production.

[0081] An additional exemplary adipate pathway utilizes alpha-ketoadipate
as a precursor (see FIG. 6 and Example VI). For example, a non-naturally
occurring microbial organism can have an adipate pathway including at
least one exogenous nucleic acid encoding an adipate pathway enzyme
expressed in a sufficient amount to produce adipate, the adipate pathway
including homocitrate synthase, homoaconitase, homoisocitrate
dehydrogenase, 2-ketoadipate reductase, alpha-hydroxyadipate dehydratase
and oxidoreductase. A further exemplary adipate pathway utilizes a lysine
dedgradation pathway (see FIG. 7 and Example VII). Another non-naturally
occurring microbial organism can have an adipate pathway including at
least one exogenous nucleic acid encoding an adipate pathway enzyme
expressed in a sufficient amount to produce adipate, the adipate pathway
including carbon nitrogen lyase, oxidoreductase, transaminase and
oxidoreductase.

[0084] In another embodiment, the invention provides a non-naturally
occurring microbial organism, including a microbial organism having a
caprolactam pathway including at least one exogenous nucleic acid
encoding a caprolactam pathway enzyme expressed in a sufficient amount to
produce caprolactam, the caprolactam pathway including
6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA synthase
(see Examples XII and XV; steps K/L of FIG. 11). Such a non-naturally
occurring microbial organism containing a caprolactam pathway can further
comprise a 6-aminocaproic acid pathway (see FIG. 11). Exemplary
6-aminocaproic acid pathways include the 6-aminocaproic acid pathway
including CoA-dependent aldehyde dehydrogenase; and transaminase or
6-aminocaproate dehydrogenase or the 6-aminocaproic acid pathway
including 3-oxo-6-aminohexanoyl-CoA thiolase;
3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoyl-CoA
synthase, or 3-oxo-6-aminohexanoyl-CoA hydrolase; 3-oxo-6-aminohexanoate
reductase; 3-hydroxy-6-aminohexanoate dehydratase; and
6-aminohex-2-enoate reductase (steps A/E/F/G/H/I/J of FIG. 11). It is
understood that these or other exemplary 6-aminocaproic acid pathways
disclosed herein can additionally be included in a microbial organism
having a caprolactam pathway, if desired. The invention also provides a
non-naturally occurring microbial organism, including a microbial
organism having a hexamethylenediamine pathway including at least one
exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme
expressed in a sufficient amount to produce hexamethylenediamine, the
hexamethylenediamine pathway including 6-aminocaproyl-CoA/acyl-CoA
transferase or 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase
(aldehyde forming); and hexamethylenediamine transaminase or
hexamethylenediamine dehydrogenase (see Example XII and XVI; steps
K/L/N/O/P of FIG. 11). Such a non-naturally occurring microbial organism
containing a hexamethylenediamine pathway can further comprise a
6-aminocaproic acid pathway (see FIG. 11). Exemplary 6-aminocaproic acid
pathways include the 6-aminocaproic acid pathway including CoA-dependent
aldehyde dehydrogenase; and transaminase or 6-aminocaproate dehydrogenase
or the 6-aminocaproic acid pathway including 3-oxo-6-aminohexanoyl-CoA
thiolase; 3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase,
3-oxo-6-aminohexanoyl-CoA synthase, or 3-oxo-6-aminohexanoyl-CoA
hydrolase; 3-oxo-6-aminohexanoate reductase; 3-hydroxy-6-aminohexanoate
dehydratase; and 6-aminohex-2-enoate reductase (steps A/E/F/G/H/I/J of
FIG. 11). It is understood that these or other exemplary 6-aminocaproic
acid pathways disclosed herein can additionally be included in a
microbial organism having a hexamethylenediamine pathway, if desired.

[0087] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
decarboxylase; or an adipate semialdehyde aminotransferase or an adipate
semialdehyde oxidoreductase (aminating) (see Examples XIX and XXI; steps
A/B/C/D/E of FIG. 12). In a further aspect, the 6-ACA pathway includes a
succinic semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase
or a phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding 6-ACA pathway enzymes, wherein the
set encodes an HODH aldolase; an OHED hydratase; an OHED reductase; a
2-OHD decarboxylase; and an adipate semialdehyde aminotransferase or an
adipate semialdehyde oxidoreductase (aminating).

[0088] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED decarboxylase; a
6-OHE reductase; or an adipate semialdehyde aminotransferase or an
adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and
XXI; steps A/B/F/G/E of FIG. 12). In a further aspect, the 6-ACA pathway
includes a succinic semialdehyde dehydrogenase, an alpha-ketoglutarate
decarboxylase or a phosphoenolpyruvate (PEP) carboxykinase. In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding 6-ACA pathway enzymes,
where the set encode an HODH aldolase; an OHED hydratase; an OHED
decarboxylase; a 6-OHE reductase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating).

[0089] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED aminotransferase
or an OHED oxidoreductase (aminating); a 2-AHE reductase; or a 2-AHD
decarboxylase (see Examples XIX and XXI; steps A/B/J/D/I of FIG. 12). In
a further aspect, the 6-ACA pathway includes a succinic semialdehyde
dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set
encode an HODH aldolase; an OHED hydratase; an OHED aminotransferase or
an OHED oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD
decarboxylase.

[0090] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
aminotransferase or a 2-OHD oxidoreductase (aminating); or a 2-AHD
decarboxylase (see Examples XIX and XXI; steps A/B/C/H/I of FIG. 12). In
a further aspect, the 6-ACA pathway includes a succinic semialdehyde
dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set
encode an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD
decarboxylase.

[0091] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an HODH formate-lyase and a pyruvate
formate-lyase activating enzyme or an HODH dehydrogenase; a
3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an
adipyl-CoA dehydrogenase; or an adipate semialdehyde aminotransferase or
an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and
XXI; steps A/L/M/N/O/E of FIG. 12). In a further aspect, the 6-ACA
pathway includes a succinic semialdehyde dehydrogenase, an
alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP)
carboxykinase. In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an
HODH formate-lyase and a pyruvate formate-lyase activating enzyme or an
HODH dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a
2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an
adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating).

[0092] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED formate-lyase and
a pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a
2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; or an
adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/E
of FIG. 12). In a further aspect, the 6-ACA pathway includes a succinic
semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase. In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the set
encode an HODH aldolase; an OHED hydratase; an OHED formate-lyase and a
pyruvate formate-lyase activating enzyme or OHED dehydrogenase; a
2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an
adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating).

[0093] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD
dehydrogenase; an adipyl-CoA dehydrogenase; or an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating)
(see Examples XIX and XXI; steps A/B/C/Q/O/E of FIG. 12). In a further
aspect, the 6-ACA pathway includes a succinic semialdehyde dehydrogenase,
an alpha-ketoglutarate decarboxylase or a phosphoenolpyruvate (PEP)
carboxykinase. In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding 6-ACA pathway enzymes, where the set encode an HODH aldolase; an
OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate
formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA
dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate
semialdehyde oxidoreductase (aminating).The invention additionally
provides a non-naturally occurring microbial organism having a
6-aminocaproic acid (6-ACA) pathway including at least one exogenous
nucleic acid encoding a 6-ACA pathway enzyme expressed in a sufficient
amount to produce 6-ACA, the 6-ACA pathway including a glutamyl-CoA
transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an
3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a
6-aminopimeloyl-CoA reductase (aldehyde forming), or a 2-aminopimelate
decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of FIG.
20). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
6-ACA pathway enzymes, where the set encode a glutamyl-CoA transferase or
glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA
oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a
6-amino-7-carboxyhept-2-enoyl-CoA reductase; 6-aminopimeloyl-CoA
reductase (aldehyde forming); and a 2-aminopimelate decarboxylase.

[0094] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase (see
Examples XXV and XXVI; steps A/B/J/T/AA of FIG. 21). In another aspect of
the invention, the non-naturally occurring microbial organism includes a
set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the
set encode a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelate
decarboxylase. The invention additionally provides a non-naturally
occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway
including at least one exogenous nucleic acid encoding a 6-ACA pathway
enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA
pathway including a homolysine 2-monooxygenase (see Examples XXV and
XXVI; steps A of FIG. 23). In a further aspect, the 6-ACA pathway
includes hydrolysis of the 6-aminohexanamide product by a dilute acid or
base to convert 6-aminohexanamide to 6-aminocaproate (see Examples XXV
and XXVI; step B of FIG. 23).

[0095] The invention additionally provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including an adipate reductase, an adipate kinase or an adipylphosphate
reductase (see Example XXVIII; steps X/Y/Z of FIG. 25 and Example XXXI).
In a further aspect, the 6-ACA pathway includes an adipate reductase. In
another further aspect, the G-ACA pathway includes an adipate kinase and
an adipylphosphate reductase. In still another aspect, the microbial
organism having the 6-aminocaproic acid (6-ACA) pathway above further
comprises an adipate pathway, a caprolactam pathway and/or a
hexamethylenediamine pathway described here (see Example)(XVIII; steps
A-W of FIG. 25).

[0096] In one embodiment, the invention provides a non-naturally occurring
microbial organism having a 6-aminocaproic acid (6-ACA) pathway including
at least one exogenous nucleic acid encoding a 6-ACA pathway enzyme
expressed in a sufficient amount to produce 6-ACA, the 6-ACA pathway
including a 2-amino-7-oxosubarate keto-acid decarboxylase, a
2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate
oxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanal
oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a
2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and
XXVI; steps A/B/D/E/F/G/I of FIG. 26). In a further aspect, the microbial
organism has a 2-amino-7-oxosubarate pathway having at least one
exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0097] In another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a
2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of FIG. 26).
In yet another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a
6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of FIG. 26).
In still yet another embodiment of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding 6-ACA pathway enzymes, where the set encodes a
2-amino-7-oxosubarate amino acid decarboxylase; a 2-amino-7-oxoheptanoate
decarboxylase; and a 6-aminohexanal oxidoreductase (see Example XXV;
steps I/G/F of FIG. 26). In a further aspect of each of the above
embodiments, the microbial organism has a 2-amino-7-oxosubarate pathway
having a second set of exogenous nucleic acids encoding
2-amino-7-oxosubarate pathway enzymes expressed in a sufficient amount to
produce 2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway
including a 2-amino-5-hydroxy-7-oxosubarate aldolase; a
2-amino-5-hydroxy-7-oxosubarate dehydratase; and a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0098] In yet another embodiment, the invention provides a non-naturally
occurring microbial organism having a hexamethylenediamine (HMDA) pathway
including at least one exogenous nucleic acid encoding a HMDA pathway
enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate kinase, an [(6-aminohexanoyl)oxy]phosphonate
(6-AHOP) oxidoreductase, a 6-aminocaproic semialdehyde aminotransferase,
a 6-aminocaproic semialdehyde oxidoreductase (aminating), a
6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate kinase, an
[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a
6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase
(aminating), a 6-acetamidohexanamine N-acetyltransferase, a
6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoA
transferase, a 6-acetamidohexanoate CoA ligase, a 6-acetamidohexanoyl-CoA
oxidoreductase, a [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP)
acyltransferase, a [(6-aminohexanoyl)oxy]phosphonate (6-AHOP)
acyltransferase, a 6-aminocaproate CoA transferase and a 6-aminocaproate
CoA ligase (see Examples XX and XXI; steps A-N of FIG. 13).

[0099] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; or a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and
XXI; steps A/B/C of FIG. 13). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
6-aminocaproate kinase; a 6-AHOP oxidoreductase; and a 6-aminocaproic
semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid
semialdehyde aminotransferase.

[0100] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate kinase; a 6-AHOP acyltransferase; a
6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic semialdehyde
oxidoreductase (aminating) or a 6-aminocaproic acid semialdehyde
aminotransferase (see Examples XX and XXI; steps A/L/N/C of FIG. 13). In
another aspect of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 6-aminocaproate kinase; a 6-AHOP
acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase.

[0101] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA
ligase; a 6-aminocaproyl-CoA oxidoreductase; or a 6-aminocaproic
semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid
semialdehyde aminotransferase (see Examples XX and XXI; steps M/N/C of
FIG. 13). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 6-aminocaproate CoA
transferase or a 6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA
oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase
(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase.

[0102] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate
kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase
or a 6-acetamidohexanal oxidoreductase (aminating); or a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide) (see Examples XX and XXI; steps D/E/F/G/H of FIG. 13).
In another aspect of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a
6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a
6-acetamidohexanal aminotransferase or a 6-acetamidohexanal
oxidoreductase (aminating); and a 6-acetamidohexanamine
N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide).

[0103] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate
CoA transferase or a 6-acetamidohexanoate CoA ligase; a
6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal
aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); or a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of FIG. 13).
In another aspect of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a
6-acetamidohexanoate CoA transferase or a 6-acetamidohexanoate CoA
ligase; a 6-acetamidohexanoyl-CoA oxidoreductase; a 6-acetamidohexanal
aminotransferase or a 6-acetamidohexanal oxidoreductase (aminating); and
a 6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide).The invention additionally provides a non-naturally
occurring microbial organism having a hexamethylenediamine (HMDA) pathway
including at least one exogenous nucleic acid encoding a HMDA pathway
enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate
kinase; a 6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase
or a 6-acetamidohexanal oxidoreductase (aminating); or a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/G of FIG. 13).
In another aspect of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 6-aminocaproate N-acetyltransferase; a
6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a
6-acetamidohexanal aminotransferase or a 6-acetamidohexanal
oxidoreductase (aminating); and a 6-acetamidohexanamine
N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide).The
invention additionally provides a non-naturally occurring microbial
organism having a hexamethylenediamine (HMDA) pathway including at least
one exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an
3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a
6-aminopimeloyl-CoA reductase (aldehyde forming), a
2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A-H of FIG. 20). In another aspect of the invention,
the non-naturally occurring microbial organism includes a set of
exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a glutamyl-CoA transferase or ligase; a beta-ketothiolase; a
3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a
6-aminopimeloyl-CoA reductase (aldehyde forming); a
2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and
a homolysine decarboxylase.

[0104] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a
3-oxo-1-carboxyheptanal aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3-oxopimelate kinase, a
5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a
3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate
kinase, a 5-aminopimeloylphosphonate reductase, a 3-aminopimelate
reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, a
homolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a
2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a
2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a
6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase
(aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a
3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and
XXVI; FIG. 21).

[0105] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a
3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S
of FIG. 21). In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a
3-oxo-1-carboxyheptanal 7-aminotransferase or a 3-oxo-1-carboxyheptanal
7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase
or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0106] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a
3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal
7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase,
a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a
3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see
Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of FIG. 21). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a
5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal
7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminating
oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a
3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0107] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a
5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal
7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/H/I/D/E/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal
7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase;
a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0108] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a
3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a
3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate
l-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/C/AB/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase or
a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a
3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate
7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0109] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a
3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a
3-oxo-1-carboxyheptanal 3-aminotransferase, a 3-oxo-1-carboxyheptanal
3-aminating oxidoreductase, a 3-amino-7-oxoheptanoate 7-aminotransferase,
a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a
3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see
Examples XXIV and XXVI; steps A/B/H/I/AB/Z/R/S of FIG. 21). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a
5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal
3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating
oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a
3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0110] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a
5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal
3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase,
a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate
7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/F/G/AB/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal
3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating
oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or
3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0111] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate
2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a
2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine
decarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of FIG.
21). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0112] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate
reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A/B/J/M/N/P/Q/S of FIG. 21). In another aspect of
the invention, the non-naturally occurring microbial organism includes a
set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the
set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate
reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0113] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a
3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate
7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or
a homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/K/L/P/Q/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate
CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a
3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate
7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase.

[0114] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase,
3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a
3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see
Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of FIG. 21). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or
3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a
3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate
l-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0115] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a
3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/K/L/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate
CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a
3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate
aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a
homolysine decarboxylase.

[0116] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a
3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/M/N/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate
reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a
3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate
2,3-aminomutase; and a homolysine decarboxylase.

[0117] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A/B/J/T/W/Q/S of FIG. 21). In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase,
a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase;
a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0118] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a
6-aminopimeloylphosphonate reductase, a 2-amino-7-oxoheptanoate
7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase,
or a homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/T/U/X/Q/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate
kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate
7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase.

[0119] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoA transferase,
2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase (aldehyde
forming), a 2-amino-7-oxoheptanoate 7-aminotransferase,
2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine
decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of FIG.
21). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or
2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde
forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or
2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine
decarboxylase.

[0120] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a
2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoate
aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating
oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate
decarboxylase, a 6-aminohexanal aminotransferase or a 6-aminohexanal
aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of FIG.
22). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a
2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a
2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate
aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase. In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a
2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate
decarboxylase; and a 6-aminohexanal aminotransferase or a 6-aminohexanal
aminating oxidoreductase.

[0121] The invention additionally provides a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde
aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase
(aminating), 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate
reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal
oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or
acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or
D/P/G/H of FIG. 24 and Example XXXI). In another aspect of the invention,
the non-naturally occurring microbial organism includes a set of
exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a 6-aminocaproate reductase; and a 6-aminocaproic semialdehyde
aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase
(aminating). In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding HMDA pathway enzymes, wherein the set encodes 6-aminocaproate
N-acetyltransferase; 6-acetamidohexanoate reductase; 6-acetamidohexanal
aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and
6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine
hydrolase (amide).The invention additionally provides a non-naturally
occurring microbial organism having a hexamethylenediamine (HMDA) pathway
including at least one exogenous nucleic acid encoding a HMDA pathway
enzyme expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 2-amino-7-oxosubarate keto-acid decarboxylase, a
2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanal aminating
oxidoreductase, a 6-aminohexanal aminotransferase, a
2-amino-7-oxoheptanoate decarboxylase, a homolysine decarboxylase, a
2-amino-7-oxosubarate amino acid decarboxylase, a 2-oxo-7-aminoheptanoate
aminating oxidoreductase, a 2-oxo-7-aminoheptanoate aminotransferase, a
2-amino-7-oxosubarate aminating oxidoreductase, a 2-amino-7-oxosubarate
aminotransferase, a 2,7-diaminosubarate decarboxylase, a
2-amino-7-oxoheptanoate aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase (see Examples XXIV and XXVI; Steps
A/B/C/G/H/I/J/K/L/M of FIG. 26). In a further aspect, the microbial
organism has a 2-amino-7-oxosubarate pathway having at least one
exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0122] In another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate
aminating oxidoreductase or 2-amino-7-oxosubarate aminotransferase; a
2,7-diaminosubarate decarboxylase; and a homolysine decarboxylase (see
Examples XXIV and XXVI; steps K/L/H of FIG. 26). In another embodiment of
the invention, the non-naturally occurring microbial organism includes a
set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the
set encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a
2-oxo-7-aminoheptanoate aminating oxidoreductase or a
2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase
(see Examples XXIV and XXVI; steps I/J/H of FIG. 26). In another
embodiment of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 2-amino-7-oxosubarate amino acid
decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a
6-aminohexanal aminating oxidoreductase or a 6-aminohexanal
aminotransferase (see Examples XXIV and XXVI; steps I/G/C of FIG. 26). In
another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a
6-aminohexanal aminating oxidoreductase or a 6-aminohexanal
aminotransferase (see Examples XXIV and XXVI; steps A/B/C of FIG. 26). In
another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating
oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a
homolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H of FIG.
26). In a further aspect of each of the above embodiments, the microbial
organism has a 2-amino-7-oxosubarate pathway having a second set of
exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase; a 2-amino-5-hydroxy-7-oxosubarate dehydratase; and a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).The invention additionally provides a non-naturally
occurring microbial organism having a levulinic acid (LA) pathway
including at least one exogenous nucleic acid encoding a LA pathway
enzyme expressed in a sufficient amount to produce LA, the LA pathway
including a 3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoA
transferase, a 3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, or
a 3-oxoadipate decarboxylase (see Example XXIX; steps A/E/F/G/AA of FIG.
25). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding LA
pathway enzymes, wherein the set encodes a 3-oxoadipyl-CoA thiolase; a
3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, or a
3-oxoadipyl-CoA hydrolase; and a 3-oxoadipate decarboxylase.

[0123] A non-naturally occurring microbial organism disclosed herein can
have, for example, a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway, wherein the non-naturally
occurring microbial organism includes at least one exogenous nucleic acid
encoding a polypeptide that converts a substrate to a product, as
disclosed herein. Thus, a non-naturally occurring microbial organism can
contain at least one exogenous nucleic acid encoding a polypeptide, where
the polypeptide is an enzyme or protein that converts the substrates and
products of a 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid pathway, such as that shown in FIGS. 2, 3, 8, 9, 10, 11,
12, 13 and 20-27.

[0124] For example, a non-naturally occurring microbial organism can have
an adipate pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from succinyl-CoA and acetyl-CoA to
3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA;
3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;
5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate (see FIG.
2). Additionally, a non-naturally occurring microbial organism can have
an adipate pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from succinyl-CoA and acetyl-CoA to
3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; 3-oxoadipate to
3-hydroxyadipate; 3-hydroxyadipate to hexa-2-enedioate (also referred to
herein as 5-carboxy-2-pentenoate); hexa-2-enedioate to adipate (see FIG.
3). Also, a non-naturally occurring microbial organism can have a
6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from adipyl-CoA to adipate semialdehyde;
and adipate semialdehyde to 6-aminocaproate (see FIG. 8). Furthermore, a
non-naturally occurring microbial organism can have a caprolactam
pathway, wherein the microbial organism contains at least one exogenous
nucleic acid encoding a polypeptide that converts a substrate to a
product selected from adipyl-CoA to adipate semialdehyde; adipate
semialdehyde to 6-aminocaproate; and 6-aminocaproate to caprolactam.
Additionally, a non-naturally occurring microbial organism can have an
adipate pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from alpha-ketoadipate to alpha-ketoadipyl-CoA;
alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to
5-carboxy-2-pentenoyl-CoA; 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and
adipyl-CoA to adipate (see FIG. 9). Also, a non-naturally occurring
microbial organism can have an adipate pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
alpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to
2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;
5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (FIG.
9).

[0125] Additionally, a non-naturally occurring microbial organism can have
a 6-aminocaproyl-CoA pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from 4-aminobutyryl-CoA and acetyl-CoA to
3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to
3-hydroxy-6-aminohexanoyl-CoA; 3-hydroxy-6-aminohexanoyl-CoA to
6-aminohex-2-enoyl-CoA; 6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA
(FIG. 11). Additional substrates and products of such a pathway can
include 6-aminocaproyl-CoA to 6-aminocaproate; 6-aminocaproyl-CoA to
caprolactam; or 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde and
6-aminocaproate semialdehyde to hexamethylenediamine (FIG. 11). A
non-naturally occurring microbial organism also can have a 6-aminocaproic
acid pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from 4-aminobutyryl-CoA and acetyl-CoA to
3-oxo-6-aminohexanoyl-CoA; 3-oxo-6-aminohexanoyl-CoA to
3-oxo-6-aminohexanoate; 3-oxo-6-aminohexanoate to
3-hydroxy-6-aminohexanoate; 3-hydroxy-6-aminohexanoate to
6-aminohex-2-enoate; and 6-aminohex-2-enoate to 6-aminocaproate (FIG.
11). Additional substrates and products of such a pathway can include
6-aminocaproate to caprolactam or 6-aminocaproate to 6-aminocaproyl-CoA,
6-aminocaproyl-CoA to 6-aminocaproate semialdehyde, and 6-aminocaproate
semialdehyde to hexamethylenediamine (FIG. 11).

[0126] Additionally, a non-naturally occurring microbial organism can have
a 6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from pyruvate and succinic semialdehyde
to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate
(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED): 2-oxohept-4-ene-1,7-dioate
(OHED) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate
(2-OHD) to adipate semialdehyde; and adipate semialdehyde to
6-aminocaproate (FIG. 12). A non-naturally occurring microbial organism
alternatively can have a 6-aminocaproic acid pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;
4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate
(OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 6-oxohex-4-enoate (6-OHE):
6-oxohex-4-enoate (6-OHE) to adipate semialdehyde; and adipate
semialdehyde to 6-aminocaproate (FIG. 12). A non-naturally occurring
microbial organism alternatively can have a 6-aminocaproic acid pathway,
wherein the microbial organism contains at least one exogenous nucleic
acid encoding a polypeptide that converts a substrate to a product
selected from pyruvate and succinic semialdehyde to
4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate
(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate
(OHED) to 2-aminohept-4-ene-1,7-dioate (2-AHE);
2-aminohept-4-ene-1,7-dioate (2-AHE) to 2-aminoheptane-1,7-dioate
(2-AHD); and 2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate (FIG.
12). A non-naturally occurring microbial organism alternatively can have
a 6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from pyruvate and succinic semialdehyde
to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate
(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate
(OHED) to 2-oxoheptane-1,7-dioate (2-OHD); 2-oxoheptane-1,7-dioate
(2-OHD) to 2-aminoheptane-1,7-dioate (2-AHD); and
2-aminoheptane-1,7-dioate (2-AHD) to 6-aminocaproate (FIG. 12). A
non-naturally occurring microbial organism alternatively can have a
6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from pyruvate and succinic semialdehyde
to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate
(HODH) to 3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA to
2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA to adipyl-CoA; adipyl-CoA to
adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (FIG.
12). A non-naturally occurring microbial organism alternatively can have
a 6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from pyruvate and succinic semialdehyde
to 4-hydroxy-2-oxoheptane-1,7-dioate; 4-hydroxy-2-oxoheptane-1,7-dioate
(HODH) to 2-oxohept-4-ene-1,7-dioate (OHED); 2-oxohept-4-ene-1,7-dioate
(OHED) to 2,3-dehydroadipyl-CoA; 2,3-dehydroadipyl-CoA to adipyl-CoA;
adipyl-CoA to adipate semialdehyde; and adipate semialdehyde to
6-aminocaproate (FIG. 12). A non-naturally occurring microbial organism
alternatively can have a 6-aminocaproic acid pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
pyruvate and succinic semialdehyde to 4-hydroxy-2-oxoheptane-1,7-dioate;
4-hydroxy-2-oxoheptane-1,7-dioate (HODH) to 2-oxohept-4-ene-1,7-dioate
(OHED); 2-oxohept-4-ene-1,7-dioate (OHED) to 2-oxoheptane-1,7-dioate
(2-OHD); 2-oxoheptane-1,7-dioate (2-OHD) to adipyl-CoA; adipyl-CoA to
adipate semialdehyde; and adipate semialdehyde to 6-aminocaproate (FIG.
12).

[0127] Additionally, a non-naturally occurring microbial organism can have
a 6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from glutamate to glutamyl-CoA;
glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA; 3-oxo-6-amino-pimeloyl-CoA to
3-hydroxy-6-amino-pimeloyl-CoA; 3-hydroxy-6-amino-pimeloyl-CoA to
6-amino-7-carboxy-hept-2-enoyl-CoA; 6-amino-7-carboxy-hept-2-enoyl-CoA to
6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to 2-aminopimelate; and
2-aminopimelate to 6-aminocaproate (FIG. 20). A non-naturally occurring
microbial organism alternatively can have a 6-aminocaproic acid pathway,
wherein the microbial organism contains at least one exogenous nucleic
acid encoding a polypeptide that converts a substrate to a product
selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to
3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to
2-aminopimelate; and 2-aminopimelate to 6-aminocaproate (FIG. 21). A
non-naturally occurring microbial organism alternatively can have a
6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from homolysine to 6-aminohexanamide; and
6-aminohexanamide to 6-aminocaproate (FIG. 23). A non-naturally occurring
microbial organism alternatively can have a 6-aminocaproic acid pathway,
wherein the microbial organism contains at least one exogenous nucleic
acid encoding a polypeptide that converts a substrate to a product
selected from adipate to adipate semialdehyde; adipate to adipylphospate;
and adipylphospate to adipate semialdehyde (FIG. 25).

[0128] Additionally, a non-naturally occurring microbial organism can have
a 6-aminocaproic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from 2-amino-7-oxosubarate to
2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal;
6-aminohexanal to 6-aminocaproate; 2-amino-7-oxosubarate to
2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to 6-aminohexanal;
2-amino-7-oxoheptanoate to 2-aminopimelate; and 2-aminopimelate to
6-aminocaproate (FIG. 26). A non-naturally occurring microbial organism
can further have a 2-amino-7-oxosubarate pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
glutamate-5-semialdehyde to 2-amino-5-hydroxy-7-oxosubarate;
2-amino-5-hydroxy-7-oxosubarate to 2-amino-5-ene-7-oxosubarate; and
2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (FIG.
27).Additionally, a non-naturally occurring microbial organism can have
an hexamethylenediamine (HMDA) pathway, wherein the microbial organism
contains at least one exogenous nucleic acid encoding a polypeptide that
converts a substrate to a product selected from 6-aminocaproate to
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP);
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproaic
semialdehyde; and 6-aminocaproaic semialdehyde to hexamethylenediamine
(FIG. 13). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from 6-aminocaproate to
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP);
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) to 6-aminocaproyl-CoA;
6-aminocaproyl-CoA to 6-aminocaproaic semialdehyde; and 6-aminocaproaic
semialdehyde to hexamethylenediamine (FIG. 13). A non-naturally occurring
microbial organism alternatively can have a HMDA pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
6-aminocaproate to 6-aminocaproyl-CoA; 6-aminocaproyl-CoA to
6-aminocaproic semialdehyde; and 6-aminocaproic semialdehyde to
hexamethylenediamine (FIG. 13). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to
[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP);
[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to 6-acetamidohexanal;
6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to
hexamethylenediamine (FIG. 13). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to
6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal;
6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to
hexamethylenediamine (FIG. 13). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
6-aminocaproate to 6-acetamidohexanoate; 6-acetamidohexanoate to
[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP);
[(6-acetamidohexanoy)oxy]phosphonate (6-AAHOP) to
6-acetamidohexanoyl-CoA; 6-acetamidohexanoyl-CoA to 6-acetamidohexanal;
6-acetamidohexanal to 6-acetamidohexanamine; and 6-acetamidohexanamine to
hexamethylenediamine (FIG. 13).

[0129] Additionally, a non-naturally occurring microbial organism can have
an hexamethylenediamine (HMDA) pathway, wherein the microbial organism
contains at least one exogenous nucleic acid encoding a polypeptide that
converts a substrate to a product selected from glutamate to
glutamyl-CoA; glutamyl-coA to 3-oxo-6-amino-pimeloyl-CoA;
3-oxo-6-amino-pimeloyl-CoA to 3-hydroxy-6-amino-pimeloyl-CoA;
3-hydroxy-6-amino-pimeloyl-CoA to 6-amino-7-carboxy-hept-2-enoyl-CoA;
6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA;
6-aminopimeloyl-CoA to 2-amino-7-oxoheptanoate; -amino-7-oxoheptanoate to
homolysine; and homolysine to HMDA (FIG. 20). A non-naturally occurring
microbial organism alternatively can have a HMDA pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-oxo-1-carboxy heptanal; 3-oxo-1-carboxy heptanal to
3-oxo-7-amino heptanoate; 3-oxo-7-amino heptanoate to 3,7-diamino
heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA
(FIG. 21). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl
phosphonate; 5-oxopimeloyl phosphonate to 3-oxo-1-carboxy heptanal;
3-oxo-1-carboxy heptanal to 3-oxo-7-amino heptanoate; 3-oxo-7-amino
heptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate to
homolysine and homolysine to HMDA (FIG. 21). A non-naturally occurring
microbial organism alternatively can have a HMDA pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 5-oxopimeloyl-CoA; 5-oxopimeloyl-CoA to 3-oxo-1-carboxy
heptanal; 3-oxo-1-carboxy heptanal to 3-oxo-7-amino heptanoate;
3-oxo-7-amino heptanoate to 3,7-diamino heptanoate; 3,7-diamino
heptanoate to homolysine and homolysine to HMDA (FIG. 21). A
non-naturally occurring microbial organism alternatively can have a HMDA
pathway, wherein the microbial organism contains at least one exogenous
nucleic acid encoding a polypeptide that converts a substrate to a
product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-oxo-1-carboxy
heptanal; 3-oxo-1-carboxy heptanal to 3-amino-7-oxoheptanoate;
3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate
to homolysine; and homolysine to HMDA (FIG. 21). A non-naturally
occurring microbial organism alternatively can have a HMDA pathway,
wherein the microbial organism contains at least one exogenous nucleic
acid encoding a polypeptide that converts a substrate to a product
selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to
3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl-CoA; 5-oxopimeloyl-CoA to
3-oxo-1-carboxy heptanal; 3-oxo-1-carboxy heptanal to
3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino
heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA
(FIG. 21). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-oxopimeloyl
phosphonate; 5-oxopimeloyl phosphonate to 3-oxo-1carboxy heptanal;
3-oxo-1-carboxy heptanal to 3-amino-7-oxoheptanoate;
3-amino-7-oxoheptanoate to 3,7-diamino heptanoate; 3,7-diamino heptanoate
to homolysine; and homolysine to HMDA (FIG. 21). A non-naturally
occurring microbial organism alternatively can have a HMDA pathway,
wherein the microbial organism contains at least one exogenous nucleic
acid encoding a polypeptide that converts a substrate to a product
selected from glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to
3-oxopimelate; 3-oxopimelate to 3-aminopimelate; 3-aminopimelate to
3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to
2-amino-7-axoheptanoate; 2-amino-7-axoheptanoate to homolysine; and
homolysine to HMDA (FIG. 21). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 5-aminopimeloyl
phosphonate; 5-aminopimeloyl phosphonate to 3-amino-7-oxoheptanoate;
3-amino-7-oxoheptanoate to 2-amino-7-axoheptanoate;
2-amino-7-axoheptanoate to homolysine; and homolysine to HMDA (FIG. 21).
A non-naturally occurring microbial organism alternatively can have a
HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 5-aminopimeloyl-CoA;
5-aminopimeloyl-CoA to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate
to 2-amino-7-axoheptanoate; 2-amino-7-axoheptanoate to homolysine; and
homolysine to HMDA (FIG. 21). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-aminopimelate; 3-aminopimelate to
3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino
heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA
(FIG. 21). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;
3-aminopimelate to 5-aminopimeloyl-CoA; 5-aminopimeloyl-CoA to
3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to 3,7-diamino
heptanoate; 3,7-diamino heptanoate to homolysine; and homolysine to HMDA
(FIG. 21). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from glutaryl-CoA to 3-oxopimeloyl-CoA;
3-oxopimeloyl-CoA to 3-oxopimelate; 3-oxopimelate to 3-aminopimelate;
3-aminopimelate to 5-aminopimeloyl phosphonate; 5-aminopimeloyl
phosphonate to 3-amino-7-oxoheptanoate; 3-amino-7-oxoheptanoate to
3,7-diamino heptanoate; 3,7-diamino heptanoate to homolysine; and
homolysine to HMDA (FIG. 21). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate;
2-aminopimelate to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to
homolysine; and homolysine to HMDA (FIG. 21). A non-naturally occurring
microbial organism alternatively can have a HMDA pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate;
2-aminopimelate to 6-aminopimeloylphosphonate; 6-aminopimeloylphosphonate
to 2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine; and
homolysine to HMDA (FIG. 21). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from
glutaryl-CoA to 3-oxopimeloyl-CoA; 3-oxopimeloyl-CoA to 3-oxopimelate;
3-oxopimelate to 3-aminopimelate; 3-aminopimelate to 2-aminopimelate;
2-aminopimelate to 6-aminopimeloyl-CoA; 6-aminopimeloyl-CoA to
2-amino-7-oxoheptanoate; 2-amino-7-oxoheptanoate to homolysine; and
homolysine to HMDA (FIG. 21). A non-naturally occurring microbial
organism alternatively can have a HMDA pathway, wherein the microbial
organism contains at least one exogenous nucleic acid encoding a
polypeptide that converts a substrate to a product selected from pyruvate
and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate; 2-oxo-4-hydroxy
7-aminoheptanoate to 2-oxo-7-amino hept-3-enoate; 2-oxo-7-amino
hept-3-enoate to 2-oxo-7-amino heptanoate; 2-oxo-7-amino heptanoate to
homolysine; and homolysine to HMDA (FIG. 22). A non-naturally occurring
microbial organism alternatively can have a HMDA pathway, wherein the
microbial organism contains at least one exogenous nucleic acid encoding
a polypeptide that converts a substrate to a product selected from
pyruvate and 4-aminobutanal to 2-oxo-4-hydroxy 7-aminoheptanoate;
2-oxo-4-hydroxy 7-aminoheptanoate to 2-oxo-7-amino hept-3-enoate;
2-oxo-7-amino hept-3-enoate to 2-oxo-7-amino heptanoate;
2-oxo-7-aminoheptanoate to 6-aminohexanal; and 6-aminohexanal to HMDA
(FIG. 22). A non-naturally occurring microbial organism alternatively can
have a HMDA pathway, wherein the microbial organism contains at least one
exogenous nucleic acid encoding a polypeptide that converts a substrate
to a product selected from 6-aminocaproate to 6-aminocaproic
semialdehyde; and 6-aminocaproic semialdehyde to HMDA (FIG. 24). A
non-naturally occurring microbial organism alternatively can have a HMDA
pathway, wherein the microbial organism contains at least one exogenous
nucleic acid encoding a polypeptide that converts a substrate to a
product selected from 6-aminocaproate to 6-acetamidohexanoate;
6-acetamidohexanoate to 6-acetamidohexanal; 6-acetamidohexanal to
6-acetamidohexanamine; 6-acetamidohexanamine to HMDA (FIG. 24). A
non-naturally occurring microbial organism alternatively can have a HMDA
pathway, wherein the microbial organism contains at least one exogenous
nucleic acid encoding a polypeptide that converts a substrate to a
product selected from 2-amino-7-oxosubarate to 2-amino-7-oxoheptanoate;
2-amino-7-oxoheptanoate to 6-aminohexanal; 6-aminohexanal to HMDA;
2-amino-7-oxosubarate to 2-oxo-7-aminoheptanoate; 2-amino-7-oxoheptanoate
to homolysine; homolysine to HMDA; 2-oxo-7-aminoheptanoate to homolysine;
2-oxo-7-aminoheptanoate to 6-aminohexanal; 2-amino-7-oxosubarate to
2,7-diaminosubarate; and 2,7-diaminosubarate to homolysine (FIG. 26). A
non-naturally occurring microbial organism can further have a
2-amino-7-oxosubarate pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from glutamate-5-semialdehyde to
2-amino-5-hydroxy-7-oxosubarate; 2-amino-5-hydroxy-7-oxosubarate to
2-amino-5-ene-7-oxosubarate; and 2-amino-5-ene-7-oxosubarate to
2-amino-7-oxosubarate (FIG. 27).

[0130] Additionally, a non-naturally occurring microbial organism can have
a levulinic acid pathway, wherein the microbial organism contains at
least one exogenous nucleic acid encoding a polypeptide that converts a
substrate to a product selected from succinyl-CoA and acetyl-CoA to
3-oxoadipyl-CoA; 3-oxoadipyl-CoA to 3-oxoadipate; and 3-oxoadipate to
levulinic acid. It is understood that any of the pathways disclosed
herein which produce an intermediate of one pathway can be used to
produce that intermediate for another pathway, if desired. For example,
as disclosed herein, the alpha-ketoadipate to adipate pathway shown in
FIG. 9 produces the intermediate adipyl-CoA, which is also an
intermediate in the pathway depicted in FIG. 10. Thus, it is understood
that an alternative pathway includes alpha-ketoadipate to adipyl-CoA,
which can be converted to adipate, 6-aminocaporate, caprolactam or
hexamethylenediamine, as depicted in FIG. 10. It is understood that any
of the pathways disclosed herein that produce a desired intermediate can
be used in combination with any other pathways disclosed herein so long
as a desired product is produced. For example, a non-naturally occurring
microbial organism disclosed herein, can have at least one nucleic acid
encoding a 6-aminocaproic acid pathway enzyme and at least one nucleic
acid encoding a hexamethylenediamine pathway enzyme, such as 2-AHD
decarboxylase (Step I of FIG. 12) and 6-acetamidohexanoate kinase (Step E
of FIG. 13), or alternatively 2-oxohept-4-ene-1,7-dioate (OHED)
decarboxylase (Step F of FIG. 12), adipate semialdehyde aminotransferase
(Step E of FIG. 12) and 6-acetamidohexanoyl-CoA oxidoreductase (Step J of
FIG. 13), or alternatively 5-carboxy-2-pentenoyl-CoA reductase (Step D of
FIG. 10), adipyl-CoA dehydrogenase (Step O of FIG. 12) and
6-aminocaproyl-CoA oxidoreductase (Step N of FIG. 13), or alternatively
2-amino-7-oxoheptanoate aminotransferase (Step G of FIG. 20) and
3,7-diaminoheptanoate 2,3-aminomutase (Step R of FIG. 21), or
alternatively 6-aminocaproate reductase (Step O of FIG. 24) and
6-aminohex-2-enoate reductase (Step J of FIG. 11), or alternatively
adipate reductase (Step X of FIG. 25) and 6-acetamidohexanoate reductase
(Step P of FIG. 24).

[0131] In an additional embodiment, the invention provides a non-naturally
occurring microbial organism having a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway, wherein the non-naturally
occurring microbial organism comprises at least one exogenous nucleic
acid encoding an enzyme or protein that converts a substrate to a product
selected from any of the substrates or products disclosed herein or shown
in any of FIGS. 1-14 and 20-27. One skilled in the art will understand
that any of the substrate-product pairs disclosed herein suitable to
produce a desired product and for which an appropriate activity is
available for the conversion of the substrate to the product can be
readily determined by one skilled in the art based on the teachings
herein. Thus, the invention provides a non-naturally occurring microbial
organism containing at least one exogenous nucleic acid encoding an
enzyme or protein, where the enzyme or protein converts the substrates
and products of a 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid pathway, such as any of those shown in FIGS. 1-14 and
20-27.

[0132] While generally described herein as a microbial organism that
contains a 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid pathway, it is understood that the invention additionally
provides a non-naturally occurring microbial organism comprising at least
one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway enzyme expressed in a
sufficient amount to produce an intermediate of a 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid pathway. For example,
as disclosed herein, 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway are exemplified in FIGS.
1-14 and 20-27. Therefore, in addition to a microbial organism containing
a 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic
acid pathway that produces 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid, the invention additionally
provides a non-naturally occurring microbial organism comprising at least
one exogenous nucleic acid encoding a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway enzyme, where the
microbial organism produces a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway intermediate, for example,
any of the intermediates shown in FIGS. 1-14 and 20-27.

[0133] It is understood that any of the pathways disclosed herein,
including those as described in the Examples and exemplified in the
Figures, including the pathways of FIGS. 1-14 and 20-27, can be utilized
to generate a non-naturally occurring microbial organism that produces
any pathway intermediate or product, as desired. As disclosed herein,
such a microbial organism that produces an intermediate can be used in
combination with another microbial organism expressing downstream pathway
enzymes to produce a desired product. However, it is understood that a
non-naturally occurring microbial organism that produces a 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid pathway
intermediate can be utilized to produce the intermediate as a desired
product.

[0134] The invention is described herein with general reference to the
metabolic reaction, reactant or product thereof, or with specific
reference to one or more nucleic acids or genes encoding an enzyme
associated with or catalyzing the referenced metabolic reaction, reactant
or product. Unless otherwise expressly stated herein, those skilled in
the art will understand that reference to a reaction also constitutes
reference to the reactants and products of the reaction. Similarly,
unless otherwise expressly stated herein, reference to a reactant or
product also references the reaction, and reference to any of these
metabolic constituents also references the gene or genes encoding the
enzymes that catalyze the referenced reaction, reactant or product.
Likewise, given the well known fields of metabolic biochemistry,
enzymology and genomics, reference herein to a gene or encoding nucleic
acid also constitutes a reference to the corresponding encoded enzyme and
the reaction it catalyzes as well as the reactants and products of the
reaction.

[0135] The non-naturally occurring microbial organisms of the invention
can be produced by introducing expressible nucleic acids encoding one or
more of the enzymes participating in one or more 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid biosynthetic
pathways. Depending on the host microbial organism chosen for
biosynthesis, nucleic acids for some or all of a particular
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
biosynthetic pathway can be expressed. For example, if a chosen host is
deficient in one or more enzymes for a desired biosynthetic pathway, then
expressible nucleic acids for the deficient enzyme(s) are introduced into
the host for subsequent exogenous expression. Alternatively, if the
chosen host exhibits endogenous expression of some pathway genes, but is
deficient in others, then an encoding nucleic acid is needed for the
deficient enzyme(s) to achieve 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid biosynthesis. Thus, a
non-naturally occurring microbial organism of the invention can be
produced by introducing exogenous enzyme activities to obtain a desired
biosynthetic pathway or a desired biosynthetic pathway can be obtained by
introducing one or more exogenous enzyme activities that, together with
one or more endogenous enzymes, produces a desired product such as
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.

[0136] Depending on the 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid biosynthetic pathway constituents
of a selected host microbial organism, the non-naturally occurring
microbial organisms of the invention will include at least one
exogenously expressed 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and
up to all encoding nucleic acids for one or more adipate, 6-aminocaproic
acid or caprolactam biosynthetic pathways. For example, 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid biosynthesis
can be established in a host deficient in a pathway enzyme through
exogenous expression of the corresponding encoding nucleic acid. In a
host deficient in all enzymes of a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway, exogenous expression of
all enzymes in the pathway can be included, although it is understood
that all enzymes of a pathway can be expressed even if the host contains
at least one of the pathway enzymes.

[0138] In the case of a 6-aminocaproic acid producing microbial organism,
exogenous expression of all enzymes in a pathway for production of
6-aminocaproic acid can be included in a host organism, such as
CoA-dependent aldehyde dehydrogenase and transaminase or CoA-dependent
aldehyde dehydrogenase and 6-aminocaproate dehydrogenase. For a
caprolactam producing microbial organism, exogenous expression of all
enzymes in a pathway for production of caprolactam can be included in a
host organism, such as CoA-dependent aldehyde dehydrogenase, transaminase
or 6-aminocaproate dehydrogenase, and amidohydrolase. In another example,
exogenous expression of all enzymes in a pathway for production of
6-aminocaproic acid (6-ACA) can be included in a host organism, such as
an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
decarboxylase; and an adipate semialdehyde aminotransferase or an adipate
semialdehyde oxidoreductase (aminating), or alternatively an HODH
aldolase; an OHED hydratase; an OHED decarboxylase; a 6-OHE reductase;
and an adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED
hydratase; an OHED aminotransferase or an OHED oxidoreductase
(aminating); a 2-AHE reductase; and a 2-AHD decarboxylase, or
alternatively an HODH aldolase; an OHED hydratase; an OHED reductase; a
2-OHD aminotransferase or a 2-OHD oxidoreductase (aminating); and a 2-AHD
decarboxylase, or alternatively an HODH aldolase; an HODH formate-lyase
and a pyruvate formate-lyase activating enzyme or an HODH dehydrogenase;
a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an
adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or
an adipate semialdehyde oxidoreductase (aminating), or alternatively an
HODH aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate
formate-lyase activating enzyme or OHED dehydrogenase; a
2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an
adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating), or alternatively an HODH aldolase; an OHED
hydratase; an OHED reductase; a 2-OHD formate-lyase and a pyruvate
formate-lyase activating enzyme or a 2-OHD dehydrogenase; an adipyl-CoA
dehydrogenase; and an adipate semialdehyde aminotransferase or an adipate
semialdehyde oxidoreductase (aminating). In a further aspect, all of the
6-ACA pathway described above can include a succinic semialdehyde
dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase. In another example, exogenous
expression of all enzymes in a pathway for production of 6-aminocaproic
acid (6-ACA) can be included in a host organism, such as a glutamyl-CoA
transferase or glutamyl-CoA ligase; a beta-ketothiolase; a
3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a
6-aminopimeloyl-CoA reductase (aldehyde forming); and a 2-aminopimelate
decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or
3-oxopimelate aminating oxidoreductase; a 3-aminopimelate
2,3-aminomutase; and a 2-aminopimelate decarboxylase.

[0139] In another example, exogenous expression of all enzymes in a
pathway for production of hexamethylenediamine can be included in a host
organism, such as a 6-aminocaproate kinase; a 6-AHOP oxidoreductase; and
a 6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase, or alternatively a
6-aminocaproate kinase; a 6-AHOP acyltransferase; a 6-aminocaproyl-CoA
oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase
(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase, or
alternatively a 6-aminocaproate CoA transferase or a 6-aminocaproate CoA
ligase; a 6-aminocaproyl-CoA oxidoreductase; and a 6-aminocaproic
semialdehyde oxidoreductase (aminating) or a 6-aminocaproic acid
semialdehyde aminotransferase, or alternatively a 6-aminocaproate
N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP
oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide), or alternatively a 6-aminocaproate
N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a
6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA
oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide), or alternatively a 6-aminocaproate
N-acetyltransferase; a 6-acetamidohexanoate kinase; a 6-AAHOP
oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide). In another example, exogenous expression of all
enzymes in a pathway for production of hexamethylenediamine can be
included in a host organism, such as a glutamyl-CoA transferase or
ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA oxidoreductase;
a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a
6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA
reductase (aldehyde forming); a 2-amino-7-oxoheptanoate aminotransferase
or aminating oxidoreductase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 7-aminotransferase
or a 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase; a
3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase, or alternatively a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a
5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal
7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminating
oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a
3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal
7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase;
a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase, or alternatively a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a
3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal
3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase
or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a
3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal
3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase
or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming), a 5-oxopimeloyl-CoA
hydrolase or a 5-oxopimeloyl-CoA ligase; a 3-oxo-1-carboxyheptanal
3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating
oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or
3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate
2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a
2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine
decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a
3-oxopimelate aminating oxidoreductase; a 3-aminopimelate kinase; a
5-aminopimeloylphosphonate reductase; a 3-amino-7-oxoheptanoate
2,3-aminomutase; a 2-amino-7-oxoheptanoate 7-aminotransferase or a
2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine
decarboxylase, or alternatively a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or a
3-oxopimelate aminating oxidoreductase; a 3-aminopimelate CoA transferase
or a 3-aminopimelate CoA ligase; a 5-aminopimeloyl-CoA reductase
(aldehyde forming); a 3-amino-7-oxoheptanoate 2,3-aminomutase; a
2-amino-7-oxoheptanoate 7-aminotransferase or 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate reductase; a 3-amino-7-oxoheptanoate
7-aminotransferase or 3-amino-7-oxoheptanoate 7-aminating oxidoreductase;
a 3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase,
or alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate
CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a
3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate
aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a
homolysine decarboxylase, or alternatively a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate kinase; a 5-aminopimeloylphosphonate reductase; a
3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate
aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a
homolysine decarboxylase, or alternatively a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase, or
alternatively a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate
kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate
7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase, or alternatively a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or
2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde
forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or
2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine
decarboxylase, or alternatively a 2-oxo-4-hydroxy-7-aminoheptanoate
aldolase; a 2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a
2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate
aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase, or alternatively a 6-aminocaproate
reductase; and a 6-aminocaproic semialdehyde aminotransferase or a
6-aminocaproic semialdehyde oxidoreductase (aminating), or alternatively
a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate reductase;
a 6-acetamidohexanal aminotransferase or 6-acetamidohexanal
oxidoreductase (aminating); and a 6-acetamidohexanamine
N-acetyltransferase or 6-acetamidohexanamine hydrolase (amide).

[0140] Depending on the 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid biosynthetic pathway constituents
of a selected host microbial organism, the non-naturally occurring
microbial organisms of the invention will include at least one
exogenously expressed 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway-encoding nucleic acid and
up to all encoding nucleic acids for one or more 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid biosynthetic
pathways. For example, 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid biosynthesis can be established in
a host deficient in a pathway enzyme or protein through exogenous
expression of the corresponding encoding nucleic acid. In a host
deficient in all enzymes or proteins of a 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid pathway, exogenous
expression of all enzyme or proteins in the pathway can be included,
although it is understood that all enzymes or proteins of a pathway can
be expressed even if the host contains at least one of the pathway
enzymes or proteins. For example, exogenous expression of all enzymes or
proteins in a pathway for production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid can be included, as disclosed
herein.

[0141] Given the teachings and guidance provided herein, those skilled in
the art will understand that the number of encoding nucleic acids to
introduce in an expressible form will, at least, parallel the adipate,
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
pathway deficiencies of the selected host microbial organism. Therefore,
a non-naturally occurring microbial organism of the invention can have at
least one, two, three, four, five, six, seven, eight, nine, ten, eleven
or twelve, up to all nucleic acids encoding the above enzymes
constituting a 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid biosynthetic pathway. In some embodiments, the
non-naturally occurring microbial organisms also can include other
genetic modifications that facilitate or optimize 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid biosynthesis or that
confer other useful functions onto the host microbial organism. One such
other functionality can include, for example, augmentation of the
synthesis of one or more of the 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway precursors such as
succinyl-CoA and/or acetyl-CoA in the case of adipate synthesis, or
adipyl-CoA or adipate in the case of 6-aminocaproic acid or caprolactam
synthesis, including the adipate pathway enzymes disclosed herein, or
pyruvate and succinic semialdehyde, glutamate, glutaryl-CoA, homolysine
or 2-amino-7-oxosubarate in the case of 6-aminocaprioate synthesis, or
6-aminocaproate, glutamate, glutaryl-CoA, pyruvate and 4-aminobutanal, or
2-amino-7-oxosubarate in the case of hexamethylenediamine synthesis.

[0142] Generally, a host microbial organism is selected such that it
produces the precursor of a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway, either as a naturally
produced molecule or as an engineered product that either provides de
novo production of a desired precursor or increased production of a
precursor naturally produced by the host microbial organism. A host
organism can be engineered to increase production of a precursor, as
disclosed herein. In addition, a microbial organism that has been
engineered to produce a desired precursor can be used as a host organism
and further engineered to express enzymes or proteins of a 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid pathway.

[0143] In some embodiments, a non-naturally occurring microbial organism
of the invention is generated from a host that contains the enzymatic
capability to synthesize 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid. In this specific embodiment it
can be useful to increase the synthesis or accumulation of a
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
pathway product to, for example, drive 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway reactions toward
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
production. Increased synthesis or accumulation can be accomplished by,
for example, overexpression of nucleic acids encoding one or more of the
above-described 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid pathway enzymes. Over expression of the 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid pathway enzyme
or enzymes can occur, for example, through exogenous expression of the
endogenous gene or genes, or through exogenous expression of the
heterologous gene or genes. Therefore, naturally occurring organisms can
be readily generated to be non-naturally occurring microbial organisms of
the invention, for example, producing 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid, through overexpression of at
least one, two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen, that is, up to all nucleic acids encoding
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
biosynthetic pathway enzymes. In addition, a non-naturally occurring
organism can be generated by mutagenesis of an endogenous gene that
results in an increase in activity of an enzyme in the 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid biosynthetic
pathway.

[0144] In particularly useful embodiments, exogenous expression of the
encoding nucleic acids is employed. Exogenous expression confers the
ability to custom tailor the expression and/or regulatory elements to the
host and application to achieve a desired expression level that is
controlled by the user. However, endogenous expression also can be
utilized in other embodiments such as by removing a negative regulatory
effector or induction of the gene's promoter when linked to an inducible
promoter or other regulatory element. Thus, an endogenous gene having a
naturally occurring inducible promoter can be up-regulated by providing
the appropriate inducing agent, or the regulatory region of an endogenous
gene can be engineered to incorporate an inducible regulatory element,
thereby allowing the regulation of increased expression of an endogenous
gene at a desired time. Similarly, an inducible promoter can be included
as a regulatory element for an exogenous gene introduced into a
non-naturally occurring microbial organism.

[0145] The invention additionally provides a non-naturally occurring
microbial organism that includes one or more gene disruptions, such as
the gene disruptions disclosed in Example XXX and Tables 14-16, where the
organism produces a 6-ACA, adipate and/or HMDA. The disruptions occur in
genes encoding an enzyme that couples production of adipate, 6-ACA and/or
HMDA to growth of the organism when the gene disruption reduces the
activity of the enzyme, such that the gene disruptions confer increased
production of adipate, 6-ACA and/or HMDA onto the non-naturally occurring
organism. Thus, the invention provides a non-naturally occurring
microbial organism, comprising one or more gene disruptions, the one or
more gene disruptions occurring in genes encoding proteins or enzymes
wherein the one or more gene disruptions confer increased production of
adipate, 6-ACA and/or HMDA in the organism. As disclosed herein, such an
organism contains a pathway for production of adipate, 6-ACA and/or HMDA,
in addition to the gene disruptions, such as those exemplified in Example
XXX and Tables 14-16.

[0146] It is understood that, in methods of the invention, any of the one
or more exogenous nucleic acids can be introduced into a microbial
organism to produce a non-naturally occurring microbial organism of the
invention. The nucleic acids can be introduced so as to confer, for
example, a 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid biosynthetic pathway onto the microbial organism.
Alternatively, encoding nucleic acids can be introduced to produce an
intermediate microbial organism having the biosynthetic capability to
catalyze some of the required reactions to confer 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid biosynthetic
capability. For example, a non-naturally occurring microbial organism
having a 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid biosynthetic pathway can comprise at least two exogenous
nucleic acids encoding desired enzymes. In the case of adipate
production, at least two exogenous nucleic acids can encode the enzymes
such as the combination of succinyl-CoA:acetyl-CoA acyl transferase and
3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyl
transferase and 3-hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoA
and 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA and
adipyl-CoA synthetase, and the like. In the case of caprolactam
production, at least two exogenous nucleic acids can encode the enzymes
such as the combination of CoA-dependent aldehyde dehydrogenase and
transaminase, or CoA-dependent aldehyde dehydrogenase and amidohydrolase,
or transaminase and amidohydrolase. In the case of 6-aminocaproic acid
production, at least two exogenous nucleic acids can encode the enzymes
such as the combination of an 4-hydroxy-2-oxoheptane-1,7-dioate (HODH)
aldolase and a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase, or a
2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a
2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, a 3-hydroxyadipyl-CoA
dehydratase and a adipyl-CoA dehydrogenase, a glutamyl-CoA transferase
and a 6-aminopimeloyl-CoA hydrolase, or a glutaryl-CoA beta-ketothiolase
and a 3-aminopimelate 2,3-aminomutase. In the case of
hexamethylenediamine production, at least two exogenous nucleic acids can
encode the enzymes such as the combination of 6-aminocaproate kinase and
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, or a
6-acetamidohexanoate kinase and an [(6-acetamidohexanoyl)oxy]phosphonate
(6-AAHOP) oxidoreductase, 6-aminocaproate N-acetyltransferase and
6-acetamidohexanoyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase and a 2-amino-7-oxoheptanoate aminotransferase, or a
3-oxopimeloyl-CoA ligase and a homolysine decarboxylase. Thus, it is
understood that any combination of two or more enzymes of a biosynthetic
pathway can be included in a non-naturally occurring microbial organism
of the invention.

[0147] Similarly, it is understood that any combination of three or more
enzymes of a biosynthetic pathway can be included in a non-naturally
occurring microbial organism of the invention, for example, in the case
of adipate production, the combination of enzymes succinyl-CoA:acetyl-CoA
acyl transferase, 3-hydroxyacyl-CoA dehydrogenase, and
3-hydroxyadipyl-CoA dehydratase; or succinyl-CoA:acetyl-CoA acyl
transferase, 3-hydroxyacyl-CoA dehydrogenase and
5-carboxy-2-pentenoyl-CoA reductase; or succinyl-CoA:acetyl-CoA acyl
transferase, 3-hydroxyacyl-CoA dehydrogenase and adipyl-CoA synthetase;
or 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and
adipyl-CoA:acetyl-CoA transferase, and so forth, as desired, so long as
the combination of enzymes of the desired biosynthetic pathway results in
production of the corresponding desired product. In the case of
6-aminocaproic acid production, the at least three exogenous nucleic
acids can encode the enzymes such as the combination of an
4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase, a
2-oxohept-4-ene-1,7-dioate (OHED) hydratase and a 2-oxoheptane-1,7-dioate
(2-OHD) decarboxylase, or a 2-oxohept-4-ene-1,7-dioate (OHED) hydratase,
a 2-aminohept-4-ene-1,7-dioate (2-AHE) reductase and a
2-aminoheptane-1,7-dioate (2-AHD) decarboxylase, or a 3-hydroxyadipyl-CoA
dehydratase, 2,3-dehydroadipyl-CoA reductase and a adipyl-CoA
dehydrogenase, or a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a
6-aminopimeloyl-CoA hydrolase and a 2-aminopimelate decarboxylase, or a
glutaryl-CoA beta-ketothiolase, a 3-aminating oxidoreductase and a
2-aminopimelate decarboxylase, or a 3-oxoadipyl-CoA thiolase, a
5-carboxy-2-pentenoate reductase and a adipate reductase. In the case of
hexamethylenediamine production, at least three exogenous nucleic acids
can encode the enzymes such as the combination of 6-aminocaproate kinase,
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase and
6-aminocaproic semialdehyde aminotransferase, or a 6-aminocaproate
N-acetyltransferase, a 6-acetamidohexanoate kinase and an
[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, or
6-aminocaproate N-acetyltransferase, a
[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) acyltransferase and
6-acetamidohexanoyl-CoA oxidoreductase, or a 3-oxo-6-aminopimeloyl-CoA
oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA dehydratase and a
homolysine decarboxylase, or a 2-oxo-4-hydroxy-7-aminoheptanoate
aldolase, a 2-oxo-7-aminohept-3-enoate reductase and a homolysine
decarboxylase, or a 6-acetamidohexanoate reductase, a 6-acetamidohexanal
aminotransferase and a 6-acetamidohexanamine N-acetyltransferase.
Similarly, any combination of four or more enzymes of a biosynthetic
pathway as disclosed herein can be included in a non-naturally occurring
microbial organism of the invention, as desired, so long as the
combination of enzymes of the desired biosynthetic pathway results in
production of the corresponding desired product.

[0148] In addition to the biosynthesis of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid as described herein,
the non-naturally occurring microbial organisms and methods of the
invention also can be utilized in various combinations with each other
and with other microbial organisms and methods well known in the art to
achieve product biosynthesis by other routes. For example, one
alternative to produce 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid other than use of the
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
producers is through addition of another microbial organism capable of
converting an adipate, 6-aminocaproic acid or caprolactam pathway
intermediate to 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid. One such procedure includes, for example, the
fermentation of a microbial organism that produces a 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid pathway intermediate.
The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic
acid pathway intermediate can then be used as a substrate for a second
microbial organism that converts the 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid pathway intermediate to
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
The 6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic
acid pathway intermediate can be added directly to another culture of the
second organism or the original culture of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid pathway intermediate
producers can be depleted of these microbial organisms by, for example,
cell separation, and then subsequent addition of the second organism to
the fermentation broth can be utilized to produce the final product
without intermediate purification steps.

[0149] In other embodiments, the non-naturally occurring microbial
organisms and methods of the invention can be assembled in a wide variety
of subpathways to achieve biosynthesis of, for example, 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid. In these
embodiments, biosynthetic pathways for a desired product of the invention
can be segregated into different microbial organisms, and the different
microbial organisms can be co-cultured to produce the final product. In
such a biosynthetic scheme, the product of one microbial organism is the
substrate for a second microbial organism until the final product is
synthesized. For example, the biosynthesis of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid can be accomplished
by constructing a microbial organism that contains biosynthetic pathways
for conversion of one pathway intermediate to another pathway
intermediate or the product. Alternatively, 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid also can be
biosynthetically produced from microbial organisms through co-culture or
co-fermentation using two organisms in the same vessel, where the first
microbial organism produces a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid intermediate and the second
microbial organism converts the intermediate to 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid.

[0150] Given the teachings and guidance provided herein, those skilled in
the art will understand that a wide variety of combinations and
permutations exist for the non-naturally occurring microbial organisms
and methods of the invention together with other microbial organisms,
with the co-culture of other non-naturally occurring microbial organisms
having subpathways and with combinations of other chemical and/or
biochemical procedures well known in the art to produce 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid.

[0151] Similarly, it is understood by those skilled in the art that a host
organism can be selected based on desired characteristics for
introduction of one or more gene disruptions to increase production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
Thus, it is understood that, if a genetic modification is to be
introduced into a host organism to disrupt a gene, any homologs,
orthologs or paralogs that catalyze similar, yet non-identical metabolic
reactions can similarly be disrupted to ensure that a desired metabolic
reaction is sufficiently disrupted. Because certain differences exist
among metabolic networks between different organisms, those skilled in
the art will understand that the actual genes disrupted in a given
organism may differ between organisms. However, given the teachings and
guidance provided herein, those skilled in the art also will understand
that the methods of the invention can be applied to any suitable host
microorganism to identify the cognate metabolic alterations needed to
construct an organism in a species of interest that will increase
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
biosynthesis. In a particular embodiment, the increased production
couples biosynthesis of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid to growth of the organism, and can
obligatorily couple production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid to growth of the organism if
desired and as disclosed herein.

[0153] In some instances, such as when a 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid biosynthetic pathway exists in an
unrelated species, 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid biosynthesis can be conferred onto the host species by,
for example, exogenous expression of a paralog or paralogs from the
unrelated species that catalyzes a similar, yet non-identical metabolic
reaction to replace the referenced reaction. Because certain differences
among metabolic networks exist between different organisms, those skilled
in the art will understand that the actual gene usage between different
organisms may differ. However, given the teachings and guidance provided
herein, those skilled in the art also will understand that the teachings
and methods of the invention can be applied to all microbial organisms
using the cognate metabolic alterations to those exemplified herein to
construct a microbial organism in a species of interest that will
synthesize 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid.

[0155] Methods for constructing and testing the expression levels of a
non-naturally occurring 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid-producing host can be performed,
for example, by recombinant and detection methods well known in the art.
Such methods can be found described in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor
Laboratory, New York (2001); and Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

[0156] Exogenous nucleic acid sequences involved in a pathway for
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid can be introduced stably or transiently into a host cell
using techniques well known in the art including, but not limited to,
conjugation, electroporation, chemical transformation, transduction,
transfection, and ultrasound transformation. For exogenous expression in
E. coli or other prokaryotic cells, some nucleic acid sequences in the
genes or cDNAs of eukaryotic nucleic acids can encode targeting signals
such as an N-terminal mitochondrial or other targeting signal, which can
be removed before transformation into prokaryotic host cells, if desired.
For example, removal of a mitochondrial leader sequence led to increased
expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338
(2005). For exogenous expression in yeast or other eukaryotic cells,
genes can be expressed in the cytosol without the addition of leader
sequence, or can be targeted to mitochondrion or other organelles, or
targeted for secretion, by the addition of a suitable targeting sequence
such as a mitochondrial targeting or secretion signal suitable for the
host cells. Thus, it is understood that appropriate modifications to a
nucleic acid sequence to remove or include a targeting sequence can be
incorporated into an exogenous nucleic acid sequence to impart desirable
properties. Furthermore, genes can be subjected to codon optimization
with techniques well known in the art to achieve optimized expression of
the proteins.

[0157] An expression vector or vectors can be constructed to include one
or more 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid biosynthetic pathway encoding nucleic acids as exemplified
herein operably linked to expression control sequences functional in the
host organism. Expression vectors applicable for use in the microbial
host organisms of the invention include, for example, plasmids, phage
vectors, viral vectors, episomes and artificial chromosomes, including
vectors and selection sequences or markers operable for stable
integration into a host chromosome. Additionally, the expression vectors
can include one or more selectable marker genes and appropriate
expression control sequences. Selectable marker genes also can be
included that, for example, provide resistance to antibiotics or toxins,
complement auxotrophic deficiencies, or supply critical nutrients not in
the culture media. Expression control sequences can include constitutive
and inducible promoters, transcription enhancers, transcription
terminators, and the like which are well known in the art. When two or
more exogenous encoding nucleic acids are to be co-expressed, both
nucleic acids can be inserted, for example, into a single expression
vector or in separate expression vectors. For single vector expression,
the encoding nucleic acids can be operationally linked to one common
expression control sequence or linked to different expression control
sequences, such as one inducible promoter and one constitutive promoter.
The transformation of exogenous nucleic acid sequences involved in a
metabolic or synthetic pathway can be confirmed using methods well known
in the art. Such methods include, for example, nucleic acid analysis such
as Northern blots or polymerase chain reaction (PCR) amplification of
mRNA, or immunoblotting for expression of gene products, or other
suitable analytical methods to test the expression of an introduced
nucleic acid sequence or its corresponding gene product. It is understood
by those skilled in the art that the exogenous nucleic acid is expressed
in a sufficient amount to produce the desired product, and it is further
understood that expression levels can be optimized to obtain sufficient
expression using methods well known in the art and as disclosed herein.

[0158] Directed evolution is one approach that involves the introduction
of mutations targeted to a specific gene in order to improve and/or alter
the properties of an enzyme. Improved and/or altered enzymes can be
identified through implementation screening assays that allow for the
identification of useful variants. Particularly useful screening methods
include sensitive high-throughput assays that allow the automated
screening of many enzyme variants (e.g., >104). Iterative rounds
of mutagenesis and screening typically are performed to identify an
enzyme with optimized properties. The greater the number of variants
screened, the higher the probability of identifying an ideally suitable
variant. Computational algorithms that can help to identify areas of the
gene for mutagenesis also have been developed and can significantly
reduce the number of enzyme variants that need to be generated and
screened.

[0159] Numerous directed evolution technologies have been developed (for
reviews, see Hibbert et al., Biomol. Eng 22:11-19 (2005); Huisman and
Lalonde, In Biocatalysis in the pharmaceutical and biotechnology
industries pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax.
Biomol.Eng 22:1-9 (2005); and Sen et al., Appl Biochem. Biotechnol
143:212-223 (2007)) to be effective at creating diverse variant libraries
and these methods have been successfully applied to the improvement of a
wide range of properties across many enzyme classes.

[0161] The following exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired properties of
specific enzymes. Any of these can be used to alter/optimize activity of
a decarboxylase enzyme.

[0162] EpPCR (Pritchard et al., J Theor. Biol 234:497-509 (2005))
introduces random point mutations by reducing the fidelity of DNA
polymerase in PCR reactions by the addition of Mn2+ ions, by biasing
dNTP concentrations, or by other conditional variations. The five step
cloning process to confine the mutagenesis to the target gene of interest
involves: 1) error-prone PCR amplification of the gene of interest; 2)
restriction enzyme digestion; 3) gel purification of the desired DNA
fragment; 4) ligation into a vector; 5) transformation of the gene
variants into a suitable host and screening of the library for improved
performance. This method can generate multiple mutations in a single gene
simultaneously, which can be useful. A high number of mutants can be
generated by EpPCR, so a high-throughput screening assay or a selection
method (especially using robotics) is useful to identify those with
desirable characteristics.

[0163] Error-prone Rolling Circle Amplification (epRCA) (Fujii et al.,
Nucleic Acids Res 32:e145 (2004); and Fujii et al., Nat. Protoc.
1:2493-2497 (2006)) has many of the same elements as epPCR except a whole
circular plasmid is used as the template and random 6-mers with
exonuclease resistant thiophosphate linkages on the last 2 nucleotides
are used to amplify the plasmid followed by transformation into cells in
which the plasmid is re-circularized at tandem repeats. Adjusting the
Mn2+ concentration can vary the mutation rate somewhat. This
technique uses a simple error-prone, single-step method to create a full
copy of the plasmid with 3-4 mutations/kbp. No restriction enzyme
digestion or specific primers are required. Additionally, this method is
typically available as a kit.

[0164] DNA or Family Shuffling (Stemmer, Proc Natl Acad Sci U.S.A.
91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994)) typically
involves digestion of two or more variant genes with nucleases such as
Dnase I or EndoV to generate a pool of random fragments that are
reassembled by cycles of annealing and extension in the presence of DNA
polymerase to create a library of chimeric genes. Fragments prime each
other and recombination occurs when one copy primes another copy
(template switch). This method can be used with >1 kbp DNA sequences.
In addition to mutational recombinants created by fragment reassembly,
this method introduces point mutations in the extension steps at a rate
similar to error-prone PCR. The method can be used to remove deleterious,
random and neutral mutations that might confer antigenicity.

[0165] Staggered Extension (StEP) (Zhao et al., Nat. Biotechnol 16:258-261
(1998)) entails template priming followed by repeated cycles of 2 step
PCR with denaturation and very short duration of annealing/extension (as
short as 5 sec). Growing fragments anneal to different templates and
extend further, which is repeated until full-length sequences are made.
Template switching means most resulting fragments have multiple parents.
Combinations of low-fidelity polymerases (Taq and Mutazyme) reduce
error-prone biases because of opposite mutational spectra.

[0166] In Random Priming Recombination (RPR) random sequence primers are
used to generate many short DNA fragments complementary to different
segments of the template. (Shao et al., Nucleic Acids Res 26:681-683
(1998)) Base misincorporation and mispriming via epPCR give point
mutations. Short DNA fragments prime one another based on homology and
are recombined and reassembled into full-length by repeated
thermocycling. Removal of templates prior to this step assures low
parental recombinants. This method, like most others, can be performed
over multiple iterations to evolve distinct properties. This technology
avoids sequence bias, is independent of gene length, and requires very
little parent DNA for the application.

[0167] In Heteroduplex Recombination linearized plasmid DNA is used to
form heteroduplexes that are repaired by mismatch repair. (Volkov et al,
Nucleic Acids Res 27:e18 (1999); and Volkov et al., Methods Enzymol.
328:456-463 (2000)) The mismatch repair step is at least somewhat
mutagenic. Heteroduplexes transform more efficiently than linear
homoduplexes. This method is suitable for large genes and whole operons.

[0168] Random Chimeragenesis on Transient Templates (RACHITT) (Coco et
al., Nat. Biotechnol 19:354-359 (2001)) employs Dnase I fragmentation and
size fractionation of ssDNA. Homologous fragments are hybridized in the
absence of polymerase to a complementary ssDNA scaffold. Any overlapping
unhybridized fragment ends are trimmed down by an exonuclease. Gaps
between fragments are filled in, and then ligated to give a pool of
full-length diverse strands hybridized to the scaffold (that contains U
to preclude amplification). The scaffold then is destroyed and is
replaced by a new strand complementary to the diverse strand by PCR
amplification. The method involves one strand (scaffold) that is from
only one parent while the priming fragments derive from other genes; the
parent scaffold is selected against. Thus, no reannealing with parental
fragments occurs. Overlapping fragments are trimmed with an exonuclease.
Otherwise, this is conceptually similar to DNA shuffling and StEP.
Therefore, there should be no siblings, few inactives, and no unshuffled
parentals. This technique has advantages in that few or no parental genes
are created and many more crossovers can result relative to standard DNA
shuffling.

[0169] Recombined Extension on Truncated templates (RETT) entails template
switching of unidirectionally growing strands from primers in the
presence of unidirectional ssDNA fragments used as a pool of templates.
(Lee et al., J. Molec. Catalysis 26:119-129 (2003)) No DNA endonucleases
are used. Unidirectional ssDNA is made by DNA polymerase with random
primers or serial deletion with exonuclease. Unidirectional ssDNA are
only templates and not primers. Random priming and exonucleases don't
introduce sequence bias as true of enzymatic cleavage of DNA
shuffling/RACHITT. RETT can be easier to optimize than StEP because it
uses normal PCR conditions instead of very short extensions.
Recombination occurs as a component of the PCR steps--no direct
shuffling. This method can also be more random than StEP due to the
absence of pauses.

[0170] In Degenerate Oligonucleotide Gene Shuffling (DOGS) degenerate
primers are used to control recombination between molecules; (Bergquist
and Gibbs, Methods Mol. Biol. 352:191-204 (2007); Bergquist et al.,
Biomol. Eng 22:63-72 (2005); Gibbs et al., Gene 271:13-20 (2001)) this
can be used to control the tendency of other methods such as DNA
shuffling to regenerate parental genes. This method can be combined with
random mutagenesis (epPCR) of selected gene segments. This can be a good
method to block the reformation of parental sequences. No endonucleases
are needed. By adjusting input concentrations of segments made, one can
bias towards a desired backbone. This method allows DNA shuffling from
unrelated parents without restriction enzyme digests and allows a choice
of random mutagenesis methods.

[0171] Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY)
creates a combinatorial library with 1 base pair deletions of a gene or
gene fragment of interest. (Ostermeier et al., Proc Natl Acad Sci U.S.A.
96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol 17:1205-1209
(1999)) Truncations are introduced in opposite direction on pieces of 2
different genes. These are ligated together and the fusions are cloned.
This technique does not require homology between the 2 parental genes.
When ITCHY is combined with DNA shuffling, the system is called SCRATCHY
(see below). A major advantage of both is no need for homology between
parental genes; for example, functional fusions between an E. coli and a
human gene were created via ITCHY. When ITCHY libraries are made, all
possible crossovers are captured.

[0172] Thio-Incremental Truncation for the Creation of Hybrid Enzymes
(THIO-ITCHY) is similar to ITCHY except that phosphothioate dNTPs are
used to generate truncations. (Lutz et al., Nucleic Acids Res 29:E16
(2001)) Relative to ITCHY, THIO-ITCHY can be easier to optimize, provide
more reproducibility, and adjustability.

[0173] SCRATCHY combines two methods for recombining genes, ITCHY and DNA
shuffling. (Lutz et al., Proc Natl Acad Sci U.S.A. 98:11248-11253 (2001))
SCRATCHY combines the best features of ITCHY and DNA shuffling. First,
ITCHY is used to create a comprehensive set of fusions between fragments
of genes in a DNA homology-independent fashion. This artificial family is
then subjected to a DNA-shuffling step to augment the number of
crossovers. Computational predictions can be used in optimization.
SCRATCHY is more effective than DNA shuffling when sequence identity is
below 80%.

[0174] In Random Drift Mutagenesis (RNDM) mutations made via epPCR
followed by screening/selection for those retaining usable activity.
(Bergquist et al., Biomol.Eng 22:63-72 (2005)) Then, these are used in
DOGS to generate recombinants with fusions between multiple active
mutants or between active mutants and some other desirable parent.
Designed to promote isolation of neutral mutations; its purpose is to
screen for retained catalytic activity whether or not this activity is
higher or lower than in the original gene. RNDM is usable in high
throughput assays when screening is capable of detecting activity above
background. RNDM has been used as a front end to DOGS in generating
diversity. The technique imposes a requirement for activity prior to
shuffling or other subsequent steps; neutral drift libraries are
indicated to result in higher/quicker improvements in activity from
smaller libraries. Though published using epPCR, this could be applied to
other large-scale mutagenesis methods.

[0175] Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis
method that: 1) generates pool of random length fragments using random
incorporation of a phosphothioate nucleotide and cleavage; this pool is
used as a template to 2) extend in the presence of "universal" bases such
as inosine; 3) replication of a inosine-containing complement gives
random base incorporation and, consequently, mutagenesis. (Wong et al.,
Biotechnol J 3:74-82 (2008); Wong et al., Nucleic Acids Res 32:e26
(2004); and Wong et al., Anal. Biochem. 341:187-189 (2005)) Using this
technique it can be possible to generate a large library of mutants
within 2-3 days using simple methods. This technique is non-directed in
comparison to the mutational bias of DNA polymerases. Differences in this
approach makes this technique complementary (or an alternative) to epPCR.

[0176] In Synthetic Shuffling, overlapping oligonucleotides are designed
to encode "all genetic diversity in targets" and allow a very high
diversity for the shuffled progeny. (Ness et al., Nat. Biotechnol
20:1251-1255 (2002)) In this technique, one can design the fragments to
be shuffled. This aids in increasing the resulting diversity of the
progeny. One can design sequence/codon biases to make more distantly
related sequences recombine at rates approaching those observed with more
closely related sequences. Additionally, the technique does not require
physically possessing the template genes.

[0177] Nucleotide Exchange and Excision Technology NexT exploits a
combination of dUTP incorporation followed by treatment with uracil DNA
glycosylase and then piperidine to perform endpoint DNA fragmentation.
(Muller et al., Nucleic Acids Res 33:e117 (2005)) The gene is reassembled
using internal PCR primer extension with proofreading polymerase. The
sizes for shuffling are directly controllable using varying dUPT::dTTP
ratios. This is an end point reaction using simple methods for uracil
incorporation and cleavage. Other nucleotide analogs, such as
8-oxo-guanine, can be used with this method. Additionally, the technique
works well with very short fragments (86 bp) and has a low error rate.
The chemical cleavage of DNA used in this technique results in very few
unshuffled clones.

[0178] In Sequence Homology-Independent Protein Recombination (SHIPREC) a
linker is used to facilitate fusion between two distantly/unrelated
genes. Nuclease treatment is used to generate a range of chimeras between
the two genes. These fusions result in libraries of single-crossover
hybrids. (Sieber et al., Nat. Biotechnol 19:456-460 (2001)) This produces
a limited type of shuffling and a separate process is required for
mutagenesis. In addition, since no homology is needed this technique can
create a library of chimeras with varying fractions of each of the two
unrelated parent genes. SHIPREC was tested with a heme-binding domain of
a bacterial CP450 fused to N-terminal regions of a mammalian CP450; this
produced mammalian activity in a more soluble enzyme.

[0179] In Gene Site Saturation Mutagenesis® (GSSM®) the starting
materials are a supercoiled dsDNA plasmid containing an insert and two
primers which are degenerate at the desired site of mutations. (Kretz et
al., Methods Enzymol. 388:3-11 (2004)) Primers carrying the mutation of
interest, anneal to the same sequence on opposite strands of DNA. The
mutation is typically in the middle of the primer and flanked on each
side by ˜20 nucleotides of correct sequence. The sequence in the
primer is NNN or NNK (coding) and MNN (noncoding) (N=all 4, K=G, T, M=A,
C). After extension, DpnI is used to digest dam-methylated DNA to
eliminate the wild-type template. This technique explores all possible
amino acid substitutions at a given locus (i.e., one codon). The
technique facilitates the generation of all possible replacements at a
single-site with no nonsense codons and results in equal to near-equal
representation of most possible alleles. This technique does not require
prior knowledge of the structure, mechanism, or domains of the target
enzyme. If followed by shuffling or Gene Reassembly, this technology
creates a diverse library of recombinants containing all possible
combinations of single-site up-mutations. The utility of this technology
combination has been demonstrated for the successful evolution of over 50
different enzymes, and also for more than one property in a given enzyme.

[0180] Combinatorial Cassette Mutagenesis (CCM) involves the use of short
oligonucleotide cassettes to replace limited regions with a large number
of possible amino acid sequence alterations. (Reidhaar-Olson et al.
Methods Enzymol. 208:564-586 (1991); and Reidhaar-Olson et al. Science
241:53-57 (1988)) Simultaneous substitutions at two or three sites are
possible using this technique. Additionally, the method tests a large
multiplicity of possible sequence changes at a limited range of sites.
This technique has been used to explore the information content of the
lambda repressor DNA-binding domain.

[0181] Combinatorial Multiple Cassette Mutagenesis (CMCM) is essentially
similar to CCM except it is employed as part of a larger program: 1) Use
of epPCR at high mutation rate to 2) ID hot spots and hot regions and
then 3) extension by CMCM to cover a defined region of protein sequence
space. (Reetz, M. T., S. Wilensek, D. Zha, and K. E. Jaeger, 2001,
Directed Evolution of an Enantioselective Enzyme through Combinatorial
Multiple-Cassette Mutagenesis. Angew. Chem. Int. Ed Engl. 40:3589-3591.)
As with CCM, this method can test virtually all possible alterations over
a target region. If used along with methods to create random mutations
and shuffled genes, it provides an excellent means of generating diverse,
shuffled proteins. This approach was successful in increasing, by
51-fold, the enantioselectivity of an enzyme.

[0182] In the Mutator Strains technique conditional is mutator plasmids
allow increases of 20- to 4000-X in random and natural mutation frequency
during selection and block accumulation of deleterious mutations when
selection is not required. (Selifonova et al., Appl Environ Microbiol
67:3645-3649 (2001)) This technology is based on a plasmid-derived mutD5
gene, which encodes a mutant subunit of DNA polymerase III. This subunit
binds to endogenous DNA polymerase III and compromises the proofreading
ability of polymerase III in any strain that harbors the plasmid. A
broad-spectrum of base substitutions and frameshift mutations occur. In
order for effective use, the mutator plasmid should be removed once the
desired phenotype is achieved; this is accomplished through a temperature
sensitive origin of replication, which allows for plasmid curing at
41° C. It should be noted that mutator strains have been explored
for quite some time (e.g., see Low et al., J. Mol. Biol. 260:359-3680
(1996)). In this technique very high spontaneous mutation rates are
observed. The conditional property minimizes non-desired background
mutations. This technology could be combined with adaptive evolution to
enhance mutagenesis rates and more rapidly achieve desired phenotypes.

[0183] "Look-Through Mutagenesis (LTM) is a multidimensional mutagenesis
method that assesses and optimizes combinatorial mutations of selected
amino acids." (Rajpal et al., Proc Natl Acad Sci U.S.A. 102:8466-8471
(2005)) Rather than saturating each site with all possible amino acid
changes, a set of nine is chosen to cover the range of amino acid R-group
chemistry. Fewer changes per site allows multiple sites to be subjected
to this type of mutagenesis. A >800-fold increase in binding affinity
for an antibody from low nanomolar to picomolar has been achieved through
this method. This method is a rational approach to minimize the number of
random combinations and can increase the ability to find improved traits
by greatly decreasing the numbers of clones to be screened. This has been
applied to antibody engineering, specifically to increase the binding
affinity and/or reduce dissociation. The technique can be combined with
either screens or selections.

[0184] Gene Reassembly is a DNA shuffling method that can be applied to
multiple genes at one time or to creating a large library of chimeras
(multiple mutations) of a single gene (Tunable GeneReassembly®
(TGR®) Technology supplied by Verenium Corporation). Typically this
technology is used in combination with ultra-high-throughput screening to
query the represented sequence space for desired improvements. This
technique allows multiple gene recombinations independent of homology.
The exact number and position of cross-over events can be pre-determined
using fragments designed via bioinformatic analysis. This technology
leads to a very high level of diversity with virtually no parental gene
reformation and a low level of inactive genes. Combined with GSSM®, a
large range of mutations can be tested for improved activity. The method
allows "blending" and "fine tuning" of DNA shuffling, e.g. codon usage
can be optimized.

[0185] In Silico Protein Design Automation (PDA) is an optimization
algorithm that anchors the structurally defined protein backbone
possessing a particular fold, and searches sequence space for amino acid
substitutions that can stabilize the fold and overall protein energetics.
(Hayes et al., Proc Natl Acad Sci U.S.A. 99:15926-15931 (2002)) This
technology uses in silico structure-based entropy predictions in order to
search for structural tolerance toward protein amino acid variations.
Statistical mechanics is applied to calculate coupling interactions at
each position. Structural tolerance toward amino acid substitution is a
measure of coupling. Ultimately, this technology is designed to yield
desired modifications of protein properties while maintaining the
integrity of structural characteristics. The method computationally
assesses and allows filtering of a very large number of possible sequence
variants (1050). The choice of sequence variants to test is related
to predictions based on the most favorable thermodynamics. Ostensibly
only stability or properties that are linked to stability can be
effectively addressed with this technology. The method has been
successfully used in some therapeutic proteins, especially in engineering
immunoglobulins. In silico predictions avoid testing extraordinarily
large numbers of potential variants. Predictions based on existing
three-dimensional structures are more likely to succeed than predictions
based on hypothetical structures. This technology can readily predict and
allow targeted screening of multiple simultaneous mutations, something
not possible with purely experimental technologies due to exponential
increases in numbers.

[0186] Iterative Saturation Mutagenesis (ISM) involves: 1) use knowledge
of structure/function to choose a likely site for enzyme improvement; 2)
saturation mutagenesis at chosen site using Stratagene QuikChange (or
other suitable means); 3) screen/select for desired properties; and 4)
with improved clone(s), start over at another site and continue
repeating. (Reetz et al., Nat. Protoc. 2:891-903 (2007); and Reetz et
al., Angew.Chem.Int.Ed Engl. 45:7745-7751 (2006)) This is a proven
methodology, which assures all possible replacements at a given position
are made for screening/selection.

[0187] Any of the aforementioned methods for mutagenesis can be used alone
or in any combination. Additionally, any one or combination of the
directed evolution methods can be used in conjunction with adaptive
evolution techniques.

[0188] The invention additionally provides methods for producing a desired
intermediate or product such as adipate, 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid. For example, a
method for producing adipate can involve culturing a non-naturally
occurring microbial organism having an adipate pathway, the pathway
including at least one exogenous nucleic acid encoding an adipate pathway
enzyme expressed in a sufficient amount to produce adipate, under
conditions and for a sufficient period of time to produce adipate, the
adipate pathway including succinyl-CoA:acetyl-CoA acyl transferase,
3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,
5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase or
phosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoA transferase
or adipyl-CoA hydrolase. Additionally, a method for producing adipate can
involve culturing a non-naturally occurring microbial organism having an
adipate pathway, the pathway including at least one exogenous nucleic
acid encoding an adipate pathway enzyme expressed in a sufficient amount
to produce adipate, under conditions and for a sufficient period of time
to produce adipate, the adipate pathway including succinyl-CoA:acetyl-CoA
acyl transferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase,
3-hydroxyadipate dehydratase, and 2-enoate reductase.

[0189] Further, a method for producing 6-aminocaproic acid can involve
culturing a non-naturally occurring microbial organism having a
6-aminocaproic acid pathway, the pathway including at least one exogenous
nucleic acid encoding a 6-aminocaproic acid pathway enzyme expressed in a
sufficient amount to produce 6-aminocaproic acid, under conditions and
for a sufficient period of time to produce 6-aminocaproic acid, the
6-aminocaproic acid pathway including CoA-dependent aldehyde
dehydrogenase and transaminase or 6-aminocaproate dehydrogenase.
Additionally, a method for producing caprolactam can involve culturing a
non-naturally occurring microbial organism having a caprolactam pathway,
the pathway including at least one exogenous nucleic acid encoding a
caprolactam pathway enzyme expressed in a sufficient amount to produce
caprolactam, under conditions and for a sufficient period of time to
produce caprolactam, the caprolactam pathway including CoA-dependent
aldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase,
and amidohydrolase.

[0190] The invention additionally provides methods for producing
6-aminocaproic acid (6-ACA) by culturing a non-naturally occurring
microbial organism having a 6-ACA pathway described herein under
conditions and for a sufficient period of time to produce 6-ACA. In one
aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an
OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating).
In another aspect, the 6-ACA pathway includes an HODH aldolase; an OHED
hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate
semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase
(aminating). In yet another aspect, the 6-ACA pathway includes an HODH
aldolase; an OHED hydratase; an OHED aminotransferase or an OHED
oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase.
In still yet another aspect, the 6-ACA pathway includes an HODH aldolase;
an OHED hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD
oxidoreductase (aminating); and a 2-AHD decarboxylase. In still yet
another aspect, the 6-ACA pathway includes an HODH aldolase; an HODH
formate-lyase and a pyruvate formate-lyase activating enzyme or an HODH
dehydrogenase; a 3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA
reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating).
In still yet another aspect, the 6-ACA pathway includes an HODH aldolase;
an OHED hydratase; an OHED formate-lyase and a pyruvate formate-lyase
activating enzyme or OHED dehydrogenase; a 2,3-dehydroadipyl-CoA
reductase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating).
In still yet another aspect, the 6-ACA pathway includes an HODH aldolase;
an OHED hydratase; an OHED reductase; a 2-OHD formate-lyase and a
pyruvate formate-lyase activating enzyme or a 2-OHD dehydrogenase; an
adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or
an adipate semialdehyde oxidoreductase (aminating). In a further aspect,
the 6-ACA pathways described above can include a succinic semialdehyde
dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase.

[0191] The invention additionally provides methods for producing
hexamethylenediamine (HMDA) by culturing a non-naturally occurring
microbial organism having a HMDA pathway described herein under
conditions and for a sufficient period of time to produce HMDA. In one
aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP
oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase
(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase. In
another aspect, the HMDA pathway includes a 6-aminocaproate kinase; a
6-AHOP acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase. In yet another aspect,
the HMDA pathway includes a 6-aminocaproate CoA transferase or a
6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase. In still yet another
aspect, the HMDA pathway includes a 6-aminocaproate N-acetyltransferase;
a 6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a
6-acetamidohexanal aminotransferase or a 6-acetamidohexanal
oxidoreductase (aminating); and a 6-acetamidohexanamine
N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide). In
still yet another aspect, the HMDA pathway includes a 6-aminocaproate
N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a
6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA
oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide). In still yet another aspect, the HMDA pathway includes
a 6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a
6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide).

[0192] Also, a method for producing adipate can involve culturing a
non-naturally occurring microbial organism having an adipate pathway, the
pathway including at least one exogenous nucleic acid encoding an adipate
pathway enzyme expressed in a sufficient amount to produce adipate, under
conditions and for a sufficient period of time to produce adipate, the
adipate pathway including alpha-ketoadipyl-CoA synthetase,
phosphotransketoadipylase/alpha-ketoadipate kinase or
alpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA
dehydrogenase; 2-hydroxyadipyl-CoA dehydratase; 5-carboxy-2-pentenoyl-CoA
reductase; and adipyl-CoA synthetase, phosphotransadipylase/adipate
kinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase.
Furthermore, a method for producing adipate can involve culturing a
non-naturally occurring microbial organism having an adipate pathway, the
pathway including at least one exogenous nucleic acid encoding an adipate
pathway enzyme expressed in a sufficient amount to produce adipate, under
conditions and for a sufficient period of time to produce adipate, the
adipate pathway including 2-hydroxyadipate dehydrogenase;
2-hydroxyadipyl-CoA synthetase,
phosphotranshydroxyadipylase/2-hydroxyadipate kinase or
2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoA
dehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA
synthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA
transferase or adipyl-CoA hydrolase.

[0194] In another embodiment, the invention provides a method for
producing caprolactam by culturing a non-naturally occurring microbial
organism having a caprolactam pathway including at least one exogenous
nucleic acid encoding a caprolactam pathway enzyme expressed in a
sufficient amount to produce caprolactam, the caprolactam pathway
including 6-aminocaproyl-CoA/acyl-CoA transferase or 6-aminocaproyl-CoA
synthase (see Examples XII and XV; steps K/L of FIG. 11). In such a
method, the caprolactam can be produced by spontaneous cyclization of
6-aminocaproyl-CoA to caprolactam (see Example XII; step Q of FIG. 11).
The invention also provides a non-naturally occurring microbial organism
having a hexamethylenediamine pathway including at least one exogenous
nucleic acid encoding a hexamethylenediamine pathway enzyme expressed in
a sufficient amount to produce hexamethylenediamine, the
hexamethylenediamine pathway including 6-aminocaproyl-CoA/acyl-CoA
transferase or 6-aminocaproyl-CoA synthase; 6-aminocaproyl-CoA reductase
(aldehyde forming); and hexamethylenediamine transaminase or
hexamethylenediamine dehydrogenase (see Examples XII and XVI; steps
K/L/N/O/P of FIG. 11).

[0195] In yet another embodiment, the invention provides a method for
producing caprolactam by culturing a non-naturally occurring microbial
organism having a caprolactam pathway including at least one exogenous
nucleic acid encoding a caprolactam pathway enzyme expressed in a
sufficient amount to produce caprolactam, the caprolactam pathway
including 3-oxo-6-aminohexanoyl-CoA thiolase; 3-oxo-6-aminohexanoyl-CoA
reductase; 3-hydroxy-6-aminohexanoyl-CoA dehydratase; and
6-aminohex-2-enoyl-CoA reductase (see Examples XII and XVII; steps
A/B/C/D of FIG. 11). In such a method, the caprolactam can be produced by
spontaneous cyclization of 6-aminocaproyl-CoA to caprolactam (see Example
XII; step Q of FIG. 11). Also provided is a method for producing
hexamethylenediamine by culturing a non-naturally occurring microbial
organism having a hexamethylenediamine pathway including at least one
exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme
expressed in a sufficient amount to produce hexamethylenediamine, the
hexamethylenediamine pathway including 3-oxo-6-aminohexanoyl-CoA
thiolase; 3-oxo-6-aminohexanoyl-CoA reductase;
3-hydroxy-6-aminohexanoyl-CoA dehydratase; 6-aminohex-2-enoyl-CoA
reductase; 6-aminocaproyl-CoA reductase (aldehyde forming); and
hexamethylenediamine transaminase or hexamethylenediamine dehydrogenase
(see Examples XII and XVIII; steps A/B/C/D/N/O/P of FIG. 11).

[0197] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-ACA pathway, the microbial
organism including at least one exogenous nucleic acid encoding a 6-ACA
pathway enzyme expressed in a sufficient amount to produce 6-ACA. In one
aspect the 6-ACA pathway includes an HODH aldolase; an OHED hydratase; an
OHED reductase; a 2-OHD decarboxylase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating)
(see Examples XIX and XXI; steps A/B/C/D/E of FIG. 12). In another aspect
of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED
hydratase; an OHED decarboxylase; a 6-OHE reductase; and an adipate
semialdehyde aminotransferase or an adipate semialdehyde oxidoreductase
(aminating) (see Examples XIX and XXI; steps A/B/F/G/E of FIG. 12). In
another aspect of the invention, the 6-ACA pathway includes an HODH
aldolase; an OHED hydratase; an OHED aminotransferase or an OHED
oxidoreductase (aminating); a 2-AHE reductase; and a 2-AHD decarboxylase
(see Examples XIX and XXI; steps A/B/J/D/I of FIG. 12). In another aspect
of the invention, the 6-ACA pathway includes an HODH aldolase; an OHED
hydratase; an OHED reductase; a 2-OHD aminotransferase or a 2-OHD
oxidoreductase (aminating); and a 2-AHD decarboxylase (see Examples XIX
and XXI; steps A/B/C/H/I of FIG. 12). In another aspect of the invention,
the 6-ACA pathway includes an HODH aldolase; an HODH formate-lyase and a
pyruvate formate-lyase activating enzyme or an HODH dehydrogenase; a
3-hydroxyadipyl-CoA dehydratase; a 2,3-dehydroadipyl-CoA reductase; an
adipyl-CoA dehydrogenase; and an adipate semialdehyde aminotransferase or
an adipate semialdehyde oxidoreductase (aminating) (see Examples XIX and
XXI; steps A/L/M/N/O/E of FIG. 12). the 6-ACA pathway includes an HODH
aldolase; an OHED hydratase; an OHED formate-lyase and a pyruvate
formate-lyase activating enzyme or OHED dehydrogenase; a
2,3-dehydroadipyl-CoA reductase; an adipyl-CoA dehydrogenase; and an
adipate semialdehyde aminotransferase or an adipate semialdehyde
oxidoreductase (aminating) (see Examples XIX and XXI; steps A/B/P/N/O/E
of FIG. 12). In another aspect of the invention, the 6-ACA pathway
includes an HODH aldolase; an OHED hydratase; an OHED reductase; a 2-OHD
formate-lyase and a pyruvate formate-lyase activating enzyme or a 2-OHD
dehydrogenase; an adipyl-CoA dehydrogenase; and an adipate semialdehyde
aminotransferase or an adipate semialdehyde oxidoreductase (aminating)
(see Examples XIX and XXI; steps A/B/C/Q/O/E of FIG. 12). In a further
aspect, the 6-ACA pathways described above can include a succinic
semialdehyde dehydrogenase, an alpha-ketoglutarate decarboxylase or a
phosphoenolpyruvate (PEP) carboxykinase.

[0198] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-ACA pathway including at least
one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a
sufficient amount to produce 6-ACA, the 6-ACA pathway including a
glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an
3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a
6-aminopimeloyl-CoA reductase (aldehyde forming), or a 2-aminopimelate
decarboxylase (see Examples XXV and XXVI; steps A/B/C/D/E/I/J of FIG.
20). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
6-ACA pathway enzymes, where the set encode a glutamyl-CoA transferase or
glutamyl-CoA ligase; a beta-ketothiolase; a 3-oxo-6-aminopimeloyl-CoA
oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA dehydratase; a
6-amino-7-carboxyhept-2-enoyl-CoA reductase; a 6-aminopimeloyl-CoA
reductase (aldehyde forming); and a 2-aminopimelate decarboxylase.

[0199] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-ACA pathway including at least
one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a
sufficient amount to produce 6-ACA, the 6-ACA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate 2,3-aminomutase, or a 2-aminopimelate decarboxylase (see
Examples XXV and XXVI; steps A/B/J/T/AA of FIG. 21). In another aspect of
the invention, the non-naturally occurring microbial organism includes a
set of exogenous nucleic acids encoding 6-ACA pathway enzymes, where the
set encode a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate 2,3-aminomutase; and a 2-aminopimelate
decarboxylase.

[0200] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-ACA pathway including at least
one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a
sufficient amount to produce 6-ACA, the 6-ACA pathway including a
homolysine 2-monooxygenase (see Examples XXV and XXVI; steps A of FIG.
23). In a further aspect, the 6-ACA pathway includes hydrolysis of the
6-aminohexanamide product by a dilute acid or base to convert
6-aminohexanamide to 6-aminocaproate (see Example XXV; steps B of FIG.
23).

[0201] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-ACA pathway including at least
one exogenous nucleic acid encoding a 6-ACA pathway enzyme expressed in a
sufficient amount to produce 6-ACA, the 6-ACA pathway including an
adipate reductase, an adipate kinase or an adipylphosphate reductase (see
Example)(XVIII; steps X/Y/Z of FIG. 25). In a further aspect, the 6-ACA
pathway includes an adipate reductase. In another further aspect, the
6-ACA pathway includes an adipate kinase and an adipylphosphate
reductase. In still another aspect, the microbial organism having the
6-aminocaproic acid (6-ACA) pathway above further comprises an adipate
pathway, a caprolactam pathway and/or a hexamethylenediamine pathway
described here (see Example)(XVIII; steps A-W of FIG. 25).

[0202] In yet another embodiment, the invention provides a method for
producing 6-aminocaproic acid (6-ACA) by culturing a non-naturally
occurring microbial organism having a 6-aminocaproic acid (6-ACA) pathway
including at least one exogenous nucleic acid encoding a 6-ACA pathway
enzyme expressed in a sufficient amount to produce 6-ACA, the 6-ACA
pathway including a 2-amino-7-oxosubarate keto-acid decarboxylase, a
2-amino-7-oxoheptanoate decarboxylase, a 2-amino-7-oxoheptanoate
oxidoreductase, a 2-aminopimelate decarboxylase, a 6-aminohexanal
oxidoreductase, a 2-amino-7-oxoheptanoate decarboxylase, or a
2-amino-7-oxosubarate amino acid decarboxylase (see Examples XXV and
XXVI; steps A/B/D/E/F/G/I of FIG. 26). In a further aspect, the microbial
organism has a 2-amino-7-oxosubarate pathway having at least one
exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0203] In another embodiment of the invention, the invention provides a
method for producing 6-aminocaproic acid (6-ACA) by culturing a
non-naturally occurring microbial organism having a 6-aminocaproic acid
(6-ACA) pathway including a set of exogenous nucleic acids encoding 6-ACA
pathway enzymes, where the set encodes a 2-amino-7-oxosubarate keto-acid
decarboxylase; a 2-amino-7-oxoheptanoate oxidoreductase; and a
2-aminopimelate decarboxylase (see Example XXV; steps A/D/E of FIG. 26).
In yet another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
6-ACA pathway enzymes, where the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a
6-aminohexanal oxidoreductase (see Example XXV; steps A/B/F of FIG. 26).
In still yet another embodiment of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding 6-ACA pathway enzymes, where the set encodes a
2-amino-7-oxosubarate amino acid decarboxylase; a 2-amino-7-oxoheptanoate
decarboxylase; and a 6-aminohexanal oxidoreductase (see Example XXV;
steps I/G/F of FIG. 26). In a further aspect of each of the above
embodiments, the microbial organism has a 2-amino-7-oxosubarate pathway
having a second set of exogenous nucleic acids encoding
2-amino-7-oxosubarate pathway enzymes expressed in a sufficient amount to
produce 2-amino-7-oxosubarate, the 2-amino-7-oxosubarate pathway
including a 2-amino-5-hydroxy-7-oxosubarate aldolase; a
2-amino-5-hydroxy-7-oxosubarate dehydratase; and a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0204] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway, the microbial
organism including at least one exogenous nucleic acid encoding a HMDA
pathway enzyme expressed in a sufficient amount to produce HMDA, the HMDA
pathway including a 6-aminocaproate kinase, an
[(6-aminohexanoyl)oxy]phosphonate (6-AHOP) oxidoreductase, a
6-aminocaproic semialdehyde aminotransferase, a 6-aminocaproic
semialdehyde oxidoreductase (aminating), a 6-aminocaproate
N-acetyltransferase, a 6-acetamidohexanoate kinase, an
[(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP) oxidoreductase, a
6-acetamidohexanal aminotransferase, a 6-acetamidohexanal oxidoreductase
(aminating), a 6-acetamidohexanamine N-acetyltransferase, a
6-acetamidohexanamine hydrolase (amide), a 6-acetamidohexanoate CoA
transferase, a 6-acetamidohexanoate CoA ligase, a 6-acetamidohexanoyl-CoA
oxidoreductase, a [(6-acetamidohexanoyl)oxy]phosphonate (6-AAHOP)
acyltransferase, a [(6-aminohexanoyl)oxy]phosphonate (6-AHOP)
acyltransferase, a 6-aminocaproate CoA transferase and a 6-aminocaproate
CoA ligase (see Examples XX and XXI; steps A-N of FIG. 13).

[0205] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway, the microbial
organism including at least one exogenous nucleic acid encoding a HMDA
pathway enzyme expressed in a sufficient amount to produce HMDA. In one
aspect the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP
oxidoreductase; and a 6-aminocaproic semialdehyde oxidoreductase
(aminating) or a 6-aminocaproic acid semialdehyde aminotransferase (see
Examples XX and XXI; steps A/B/C of FIG. 13). In another aspect of the
invention, the HMDA pathway includes a 6-aminocaproate kinase; a 6-AHOP
acyltransferase; a 6-aminocaproyl-CoA oxidoreductase; and a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and
XXI; steps A/L/N/C of FIG. 13). In another aspect of the invention, the
HMDA pathway includes a 6-aminocaproate CoA transferase or a
6-aminocaproate CoA ligase; a 6-aminocaproyl-CoA oxidoreductase; and a
6-aminocaproic semialdehyde oxidoreductase (aminating) or a
6-aminocaproic acid semialdehyde aminotransferase (see Examples XX and
XXI; steps M/N/C of FIG. 13). In another aspect of the invention, the
HMDA pathway includes a 6-aminocaproate N-acetyltransferase; a
6-acetamidohexanoate kinase; a 6-AAHOP oxidoreductase; a
6-acetamidohexanal aminotransferase or a 6-acetamidohexanal
oxidoreductase (aminating); and a 6-acetamidohexanamine
N-acetyltransferase or a 6-acetamidohexanamine hydrolase (amide) (see
Examples XX and XXI; steps D/E/F/G/H of FIG. 13). In another aspect of
the invention, the HMDA pathway includes a 6-aminocaproate
N-acetyltransferase; a 6-acetamidohexanoate CoA transferase or a
6-acetamidohexanoate CoA ligase; a 6-acetamidohexanoyl-CoA
oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide) (see Examples XX and XXI; steps D/I/J/G/H of FIG. 13).
In another aspect of the invention, the HMDA pathway includes a
6-aminocaproate N-acetyltransferase; a 6-acetamidohexanoate kinase; a
6-AAHOP oxidoreductase; a 6-acetamidohexanal aminotransferase or a
6-acetamidohexanal oxidoreductase (aminating); and a
6-acetamidohexanamine N-acetyltransferase or a 6-acetamidohexanamine
hydrolase (amide) (see Examples XX and XXI; steps D/E/K/J/G of FIG. 13).

[0206] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutamyl-CoA transferase, a glutamyl-CoA ligase, a beta-ketothiolase, an
3-oxo-6-aminopimeloyl-CoA oxidoreductase, a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase, a 6-amino-7-carboxyhept-2-enoyl-CoA reductase, a
6-aminopimeloyl-CoA reductase (aldehyde forming), a
2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A-H of FIG. 20). In another aspect of the invention,
the non-naturally occurring microbial organism includes a set of
exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a glutamyl-CoA transferase or ligase; a beta-ketothiolase; a
3-oxo-6-aminopimeloyl-CoA oxidoreductase; a 3-hydroxy-6-aminopimeloyl-CoA
dehydratase; a 6-amino-7-carboxyhept-2-enoyl-CoA reductase; a
6-aminopimeloyl-CoA reductase (aldehyde forming); a
2-amino-7-oxoheptanoate aminotransferase or aminating oxidoreductase; and
a homolysine decarboxylase.

[0207] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal aminotransferase, a
3-oxo-1-carboxyheptanal aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3-oxopimelate kinase, a
5-oxopimeloylphosphonate reductase, a 3-oxopimelate CoA transferase, a
3-oxopimelate ligase, a 5-oxopimeloyl-CoA reductase (aldehyde forming), a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a 3-aminopimelate
kinase, a 5-aminopimeloylphosphonate reductase, a 3-aminopimelate
reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, a
homolysine decarboxylase, a 3-aminopimelate 2,3-aminomutase, a
2-aminopimelate kinase, a 2-aminopimelate CoA transferase, a
2-aminopimelate CoA ligase, a 2-aminopimelate reductase, a
6-aminopimeloylphosphonate reductase, a 6-aminopimeloyl-CoA reductase
(aldehyde forming), a 3-amino-7-oxoheptanoate 7-aminotransferase or a
3-amino-7-oxoheptanoate aminating oxidoreductase (see Examples XXIV and
XXVI; FIG. 21).

[0208] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 7-aminotransferase, a
3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps A/B/C/D/E/R/S
of FIG. 21). In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate reductase; a
3-oxo-1-carboxyheptanal 7-aminotransferase or a 3-oxo-1-carboxyheptanal
7-aminating oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase
or a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0209] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate kinase, a 5-oxopimeloylphosphonate reductase, a
3-oxo-1-carboxyheptanal 7-aminotransferase, a 3-oxo-1-carboxyheptanal
7-aminating oxidoreductase, a 3-oxo-7-aminoheptanoate 3-aminotransferase,
a 3-oxo-7-aminoheptanoate 3-aminating oxidoreductase, a
3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see
Examples XXIV and XXVI; steps A/B/F/G/D/E/R/S of FIG. 21). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate kinase; a
5-oxopimeloylphosphonate reductase; a 3-oxo-1-carboxyheptanal
7-aminotransferase or a 3-oxo-1-carboxyheptanal 7-aminating
oxidoreductase; a 3-oxo-7-aminoheptanoate 3-aminotransferase or a
3-oxo-7-aminoheptanoate 3-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0210] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate CoA transferase, 3-oxopimelate CoA ligase, a
5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal
7-aminotransferase, 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase, a
3-oxo-7-aminoheptanoate 3-aminotransferase, a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/H/I/D/E/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate CoA transferase or 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal
7-aminotransferase or 3-oxo-1-carboxyheptanal 7-aminating oxidoreductase;
a 3-oxo-7-aminoheptanoate 3-aminotransferase or a 3-oxo-7-aminoheptanoate
3-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0211] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate reductase, a 3-oxo-1-carboxyheptanal 3-aminotransferase, a
3-oxo-1-carboxyheptanal 3-aminating oxidoreductase, a
3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate
7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/C/AB/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate reductase; a 3-oxo-1-carboxyheptanal 3-aminotransferase or
a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase; a
3-amino-7-oxoheptanoate 7-aminotransferase or a 3-amino-7-oxoheptanoate
7-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0212] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, 3-oxopimeloyl-CoA ligase, a 3-oxopimelate
kinase, a 5-oxopimeloylphosphonate reductase, a 3-oxo-1-carboxyheptanal
3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase,
a 3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate
7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/H/I/AB/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate kinase; a 5-oxopimeloylphosphonate reductase; a
3-oxo-1-carboxyheptanal 3-aminotransferase or a 3-oxo-1-carboxyheptanal
3-aminating oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase
or a 3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0213] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase, a
5-oxopimeloyl-CoA reductase (aldehyde forming), a 3-oxo-1-carboxyheptanal
3-aminotransferase, a 3-oxo-1-carboxyheptanal 3-aminating oxidoreductase,
a 3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate
7-aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/F/G/AB/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate CoA transferase or a 3-oxopimelate CoA ligase; a
5-oxopimeloyl-CoA reductase (aldehyde forming); a 3-oxo-1-carboxyheptanal
3-aminotransferase or a 3-oxo-1-carboxyheptanal 3-aminating
oxidoreductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or
3-amino-7-oxoheptanoate 7-aminating oxidoreductase; a
3,7-diaminoheptanoate 2,3-aminomutase; and a homolysine decarboxylase.

[0214] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase, a 3-aminopimelate reductase, a 3-amino-7-oxoheptanoate
2,3-aminomutase, a 2-amino-7-oxoheptanoate 7-aminotransferase, a
2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine
decarboxylase (see Examples XXIV and XXVI; steps A/B//J/O/P/Q/S of FIG.
21). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0215] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase, a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate
reductase, a 3-amino-7-oxoheptanoate 2,3-aminomutase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A/B/J/M/N/P/Q/S of FIG. 21). In another aspect of
the invention, the non-naturally occurring microbial organism includes a
set of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the
set encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA
hydrolase, a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase;
a 3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate
reductase; a 3-amino-7-oxoheptanoate 2,3-aminomutase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0216] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a
3-amino-7-oxoheptanoate 2,3-aminomutase, a 2-amino-7-oxoheptanoate
7-aminotransferase, 2-amino-7-oxoheptanoate aminating oxidoreductase, or
a homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/K/L/P/Q/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate
CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a
3-amino-7-oxoheptanoate 2,3-aminomutase; a 2-amino-7-oxoheptanoate
7-aminotransferase or 2-amino-7-oxoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase.

[0217] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate reductase, a 3-amino-7-oxoheptanoate 7-aminotransferase,
3-amino-7-oxoheptanoate 7-aminating oxidoreductase, a
3,7-diaminoheptanoate 2,3-aminomutase, or a homolysine decarboxylase (see
Examples XXIV and XXVI; steps A/B/J/O/Z/R/S of FIG. 21). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a glutaryl-CoA beta-ketothiolase; a
3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA transferase or a
3-oxopimeloyl-CoA ligase; a 3-oxopimelate aminotransferase or
3-oxopimelate aminating oxidoreductase; a 3-aminopimelate reductase; a
3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate
l-aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and
a homolysine decarboxylase.

[0218] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate CoA transferase, a 3-aminopimelate CoA ligase, a
5-aminopimeloyl-CoA reductase (aldehyde forming), a
3-amino-7-oxoheptanoate 7-aminotransferase, 3-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/K/L/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate CoA transferase or a 3-aminopimelate
CoA ligase; a 5-aminopimeloyl-CoA reductase (aldehyde forming); a
3-amino-7-oxoheptanoate 7-aminotransferase or 3-amino-7-oxoheptanoate
aminating oxidoreductase; a 3,7-diaminoheptanoate 2,3-aminomutase; and a
homolysine decarboxylase.

[0219] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate kinase, a 5-aminopimeloylphosphonate reductase, a
3-amino-7-oxoheptanoate 7-aminotransferase, a 3-amino-7-oxoheptanoate
aminating oxidoreductase, a 3,7-diaminoheptanoate 2,3-aminomutase, or a
homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/M/N/Z/R/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate kinase; a 5-aminopimeloylphosphonate
reductase; a 3-amino-7-oxoheptanoate 7-aminotransferase or a
3-amino-7-oxoheptanoate aminating oxidoreductase; a 3,7-diaminoheptanoate
2,3-aminomutase; and a homolysine decarboxylase.

[0220] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, 3-oxopimelate aminating oxidoreductase, a
3-aminopimelate 2,3-aminomutase, a 2-aminopimelate reductase, a
2-amino-7-oxoheptanoate 7-aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, or a homolysine decarboxylase (see Examples
XXIV and XXVI; steps A/B/J/T/W/Q/S of FIG. 21). In another aspect of the
invention, the non-naturally occurring microbial organism includes a set
of exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase,
a 3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or 3-oxopimelate aminating oxidoreductase;
a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate reductase; a
2-amino-7-oxoheptanoate 7-aminotransferase or a 2-amino-7-oxoheptanoate
aminating oxidoreductase; and a homolysine decarboxylase.

[0221] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate kinase, a
6-aminopimeloylphosphonate reductase, a 2-amino-7-oxoheptanoate
7-aminotransferase, a 2-amino-7-oxoheptanoate aminating oxidoreductase,
or a homolysine decarboxylase (see Examples XXIV and XXVI; steps
A/B/J/T/U/X/Q/S of FIG. 21). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
glutaryl-CoA beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase or a 3-oxopimeloyl-CoA ligase; a
3-oxopimelate aminotransferase or a 3-oxopimelate aminating
oxidoreductase; a 3-aminopimelate 2,3-aminomutase; a 2-aminopimelate
kinase; a 6-aminopimeloylphosphonate reductase; a 2-amino-7-oxoheptanoate
7-aminotransferase or a 2-amino-7-oxoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase.

[0222] In yet another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a HMDA pathway including at least one
exogenous nucleic acid encoding a HMDA pathway enzyme expressed in a
sufficient amount to produce HMDA, the HMDA pathway including a
glutaryl-CoA beta-ketothiolase, a 3-oxopimeloyl-CoA hydrolase, a
3-oxopimeloyl-CoA transferase, a 3-oxopimeloyl-CoA ligase, a
3-oxopimelate aminotransferase, a 3-oxopimelate aminating oxidoreductase,
a 3-aminopimelate 2,3-aminomutase, a 2-aminopimelate CoA transferase,
2-aminopimelate CoA ligase, a 6-aminopimeloyl-CoA reductase (aldehyde
forming), a 2-amino-7-oxoheptanoate 7-aminotransferase,
2-amino-7-oxoheptanoate aminating oxidoreductase, or a homolysine
decarboxylase (see Examples XXIV and XXVI; steps A/B/J/T/V/Y/Q/S of FIG.
21). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a glutaryl-CoA
beta-ketothiolase; a 3-oxopimeloyl-CoA hydrolase, a 3-oxopimeloyl-CoA
transferase or a 3-oxopimeloyl-CoA ligase; a 3-oxopimelate
aminotransferase or a 3-oxopimelate aminating oxidoreductase; a
3-aminopimelate 2,3-aminomutase; a 2-aminopimelate CoA transferase or
2-aminopimelate CoA ligase; a 6-aminopimeloyl-CoA reductase (aldehyde
forming); a 2-amino-7-oxoheptanoate 7-aminotransferase or
2-amino-7-oxoheptanoate aminating oxidoreductase; and a homolysine
decarboxylase.

[0223] The invention additionally provides a method for producing
hexamethylenediamine (HMDA) by culturing a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 2-oxo-4-hydroxy-7-aminoheptanoate aldolase, a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, a
2-oxo-7-aminohept-3-enoate reductase, a 2-oxo-7-aminoheptanoate
aminotransferase, a 2-oxo-7-aminoheptanoate aminotransferase aminating
oxidoreductase, a homolysine decarboxylase, a 2-oxo-7-aminoheptanoate
decarboxylase, a 6-aminohexanal aminotransferase or 6-aminohexanal
aminating oxidoreductase (see Examples XXIV and XXVI; steps A-G of FIG.
22). In another aspect of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a
2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a
2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate
aminotransferase or a 2-oxo-7-aminoheptanoate aminating oxidoreductase;
and a homolysine decarboxylase. In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
2-oxo-4-hydroxy-7-aminoheptanoate aldolase; a
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase; a
2-oxo-7-aminohept-3-enoate reductase; a 2-oxo-7-aminoheptanoate
decarboxylase; and a 6-aminohexanal aminotransferase or a 6-aminohexanal
aminating oxidoreductase.

[0224] The invention additionally provides a method for producing
hexamethylenediamine (HMDA) by culturing a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 6-aminocaproate reductase, a 6-aminocaproic semialdehyde
aminotransferase, a 6-aminocaproic semialdehyde oxidoreductase
(aminating), 6-aminocaproate N-acetyltransferase, a 6-acetamidohexanoate
reductase, 6-acetamidohexanal aminotransferase, 6-acetamidohexanal
oxidoreductase (aminating), 6-acetamidohexanamine N-acetyltransferase or
acetamidohexanamine hydrolase (amide) (see Example XXVII; steps O/C or
D/P/G/H of FIG. 24). In another aspect of the invention, the
non-naturally occurring microbial organism includes a set of exogenous
nucleic acids encoding HMDA pathway enzymes, wherein the set encodes a
6-aminocaproate reductase; and a 6-aminocaproic semialdehyde
aminotransferase or a 6-aminocaproic semialdehyde oxidoreductase
(aminating). In another aspect of the invention, the non-naturally
occurring microbial organism includes a set of exogenous nucleic acids
encoding HMDA pathway enzymes, wherein the set encodes 6-aminocaproate
N-acetyltransferase; 6-acetamidohexanoate reductase; 6-acetamidohexanal
aminotransferase or 6-acetamidohexanal oxidoreductase (aminating); and
6-acetamidohexanamine N-acetyltransferase or 6-acetamidohexanamine
hydrolase (amide).

[0225] The invention additionally provides a method for producing
hexamethylenediamine (HMDA) by culturing a non-naturally occurring
microbial organism having a hexamethylenediamine (HMDA) pathway including
at least one exogenous nucleic acid encoding a HMDA pathway enzyme
expressed in a sufficient amount to produce HMDA, the HMDA pathway
including a 2-amino-7-oxosubarate keto-acid decarboxylase, a
2-amino-7-oxoheptanoate decarboxylase, a 6-aminohexanal aminating
oxidoreductase, a 6-aminohexanal aminotransferase, a
2-amino-7-oxoheptanoate aminotransferase, a 2-amino-7-oxoheptanoate
aminating oxidoreductase, a 2-oxo-7-aminoheptanoate decarboxylase, a
homolysine decarboxylase, a 2-amino-7-oxosubarate amino acid
decarboxylase, a 2-oxo-7-aminoheptanoate aminating oxidoreductase, a
2-oxo-7-aminoheptanoate aminotransferase, a 2-amino-7-oxosubarate
aminating oxidoreductase, a 2-amino-7-oxosubarate aminotransferase or a
2,7-diaminosubarate decarboxylase (see Examples XXIV and XXVI; Steps
A/B/C/G/H/I/J/K/L/M of FIG. 26). In a further aspect, the microbial
organism has a 2-amino-7-oxosubarate pathway having at least one
exogenous nucleic acid encoding a 2-amino-7-oxosubarate pathway enzyme
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase, a 2-amino-5-hydroxy-7-oxosubarate dehydratase, or a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0226] In another embodiment, the invention provides a method for
producing hexamethylenediamine (HMDA) by culturing a non-naturally
occurring microbial organism having a hexamethylenediamine (HMDA) pathway
including a set of exogenous nucleic acids encoding HMDA pathway enzymes,
wherein the set encodes a 2-amino-7-oxosubarate aminating oxidoreductase
or 2-amino-7-oxosubarate aminotransferase; a 2,7-diaminosubarate
decarboxylase; and a homolysine decarboxylase (see Examples XXIV and
XXVI; steps K/L/H of FIG. 26). In another embodiment of the invention,
the non-naturally occurring microbial organism includes a set of
exogenous nucleic acids encoding HMDA pathway enzymes, wherein the set
encodes a 2-amino-7-oxosubarate amino acid decarboxylase; a
2-oxo-7-aminoheptanoate aminating oxidoreductase or a
2-oxo-7-aminoheptanoate aminotransferase; and a homolysine decarboxylase
(see Examples XXIV and XXVI; steps I/J/H of FIG. 26). In another
embodiment of the invention, the non-naturally occurring microbial
organism includes a set of exogenous nucleic acids encoding HMDA pathway
enzymes, wherein the set encodes a 2-amino-7-oxosubarate amino acid
decarboxylase; a 2-oxo-7-aminoheptanoate decarboxylase; and a
6-aminohexanal aminating oxidoreductase or a 6-aminohexanal
aminotransferase (see Examples XXIV and XXVI; steps I/G/C of FIG. 26). In
another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate decarboxylase; and a
6-aminohexanal aminating oxidoreductase or a 6-aminohexanal
aminotransferase (see Examples XXIV and XXVI; steps A/B/C of FIG. 26). In
another embodiment of the invention, the non-naturally occurring
microbial organism includes a set of exogenous nucleic acids encoding
HMDA pathway enzymes, wherein the set encodes a 2-amino-7-oxosubarate
keto-acid decarboxylase; a 2-amino-7-oxoheptanoate aminating
oxidoreductase or a 2-amino-7-oxoheptanoate aminotransferase; and a
homolysine decarboxylase (see Examples XXIV and XXVI; steps A/M/H of FIG.
26). In a further aspect of each of the above embodiments, the microbial
organism has a 2-amino-7-oxosubarate pathway having a second set of
exogenous nucleic acids encoding 2-amino-7-oxosubarate pathway enzymes
expressed in a sufficient amount to produce 2-amino-7-oxosubarate, the
2-amino-7-oxosubarate pathway including a 2-amino-5-hydroxy-7-oxosubarate
aldolase; a 2-amino-5-hydroxy-7-oxosubarate dehydratase; and a
2-amino-5-ene-7-oxosubarate reductase (see Examples XXV and XXVI; steps
A/B/C of FIG. 27).

[0227] The invention additionally provides a method for producing
hexamethylenediamine (HMDA) by culturing a non-naturally occurring
microbial organism having a levulinic acid (LA) pathway including at
least one exogenous nucleic acid encoding a LA pathway enzyme expressed
in a sufficient amount to produce LA, the LA pathway including a
3-oxoadipyl-CoA thiolase, a 3-oxoadipyl-CoA/acyl-CoA transferase, a
3-oxoadipyl-CoA synthase, a 3-oxoadipyl-CoA hydrolase, or a 3-oxoadipate
decarboxylase (see Example XXIX; steps A/E/F/G/AA of FIG. 25). In another
aspect of the invention, the non-naturally occurring microbial organism
includes a set of exogenous nucleic acids encoding LA pathway enzymes,
wherein the set encodes a 3-oxoadipyl-CoA thiolase; a
3-oxoadipyl-CoA/acyl-CoA transferase, a 3-oxoadipyl-CoA synthase, or a
3-oxoadipyl-CoA hydrolase; and a 3-oxoadipate decarboxylase.

[0228] The invention further provides methods of producing non-naturally
microbial organisms having increased production of adipate, 6-ACA and/or
HMDA by disruption of one or more genes to confer increased production of
adiate, 6-ACA and/or HMDA. Such gene disruptions include those
exemplified herein in Example XXX and Tables 14-16.

[0229] The invention additionally provides a method for producing adipate,
6-ACA and/or HMDA that includes culturing a non-naturally occurring
microbial organism that includes one or more gene disruptions that confer
increased production of adiapte, 6-ACA and/or HMDA. The disruptions can
occur in genes encoding an enzyme obligatory to coupling adipate, 6-ACA
and/or HMDA production to growth of the microorganism when the gene
disruption reduces an activity of the enzyme, such that the disruptions
confer stable growth-coupled production of adipate, 6-ACA and/or HMDA
onto the non-naturally microbial organism.

[0230] In some embodiments, the gene disruption can include a complete
gene deletion. Methods for gene disruption are well known to those
skilled in the art and are described herein (see Example XXX). In some
embodiments other methods to disrupt a gene include, for example,
frameshifting by omission, addition of oligonucleotides or by mutations
that render the gene inoperable. One skilled in the art will recognize
the advantages of gene deletions, however, because of the stability it
can confer to the non-naturally occurring organism from reverting to a
phenotype expressing the previously disrupted gene. In particular, the
gene disruptions are selected from the gene sets that described in Tables
14-16.

[0231] Suitable purification and/or assays to test for the production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
can be performed using well known methods. Suitable replicates such as
triplicate cultures can be grown for each engineered strain to be tested.
For example, product and byproduct formation in the engineered production
host can be monitored. The final product and intermediates, and other
organic compounds, can be analyzed by methods such as HPLC (High
Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass
Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or
other suitable analytical methods using routine procedures well known in
the art. The release of product in the fermentation broth can also be
tested with the culture supernatant. Byproducts and residual glucose can
be quantified by HPLC using, for example, a refractive index detector for
glucose and alcohols, and a UV detector for organic acids (Lin et al.,
Biotechnol. Bioeng. 90:775-779 (2005)), or other suitable assay and
detection methods well known in the art. The individual enzyme activities
from the exogenous DNA sequences can also be assayed using methods well
known in the art.

[0232] The 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid can be separated from other components in the culture
using a variety of methods well known in the art. Such separation methods
include, for example, extraction procedures as well as methods that
include continuous liquid-liquid extraction, pervaporation, membrane
filtration, membrane separation, reverse osmosis, electrodialysis,
distillation, crystallization, centrifugation, extractive filtration, ion
exchange chromatography, size exclusion chromatography, adsorption
chromatography, and ultrafiltration. All of the above methods are well
known in the art.

[0233] Any of the non-naturally occurring microbial organisms described
herein can be cultured to produce and/or secrete the biosynthetic
products of the invention. For example, the 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid producers can be
cultured for the biosynthetic production of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid.

[0234] For the production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid, the recombinant strains are
cultured in a medium with carbon source and other essential nutrients. It
is sometimes desirable and can be highly desirable to maintain anaerobic
conditions in the fermenter to reduce the cost of the overall process.
Such conditions can be obtained, for example, by first sparging the
medium with nitrogen and then sealing the flasks with a septum and
crimp-cap. For strains where growth is not observed anaerobically,
microaerobic or substantially anaerobic conditions can be applied by
perforating the septum with a small hole for limited aeration. Exemplary
anaerobic conditions have been described previously and are well-known in
the art. Exemplary aerobic and anaerobic conditions are described, for
example, in United State publication 2009/0047719 (Ser. No. 11/891,602),
filed Aug. 10, 2007. Fermentations can be performed in a batch, fed-batch
or continuous manner, as disclosed herein.

[0235] If desired, the pH of the medium can be maintained at a desired pH,
in particular neutral pH, such as a pH of around 7 by addition of a base,
such as NaOH or other bases, or acid, as needed to maintain the culture
medium at a desirable pH. The growth rate can be determined by measuring
optical density using a spectrophotometer (600 nm), and the glucose
uptake rate by monitoring carbon source depletion over time.

[0236] The growth medium can include, for example, any carbohydrate source
which can supply a source of carbon to the non-naturally occurring
microorganism. Such sources include, for example, sugars such as glucose,
xylose, arabinose, galactose, mannose, fructose, sucrose and starch.
Other sources of carbohydrate include, for example, renewable feedstocks
and biomass. Exemplary types of biomasses that can be used as feedstocks
in the methods of the invention include cellulosic biomass,
hemicellulosic biomass and lignin feedstocks or portions of feedstocks.
Such biomass feedstocks contain, for example, carbohydrate substrates
useful as carbon sources such as glucose, xylose, arabinose, galactose,
mannose, fructose and starch. Given the teachings and guidance provided
herein, those skilled in the art will understand that renewable
feedstocks and biomass other than those exemplified above also can be
used for culturing the microbial organisms of the invention for the
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid.

[0237] In addition to renewable feedstocks such as those exemplified
above, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid microbial organisms of the invention also can be modified
for growth on syngas as its source of carbon. In this specific
embodiment, one or more proteins or enzymes are expressed in the
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
producing organisms to provide a metabolic pathway for utilization of
syngas or other gaseous carbon source.

[0238] Synthesis gas, also known as syngas or producer gas, is the major
product of gasification of coal and of carbonaceous materials such as
biomass materials, including agricultural crops and residues. Syngas is a
mixture primarily of H2 and CO and can be obtained from the
gasification of any organic feedstock, including but not limited to coal,
coal oil, natural gas, biomass, and waste organic matter. Gasification is
generally carried out under a high fuel to oxygen ratio. Although largely
H2 and CO, syngas can also include CO2 and other gases in
smaller quantities. Thus, synthesis gas provides a cost effective source
of gaseous carbon such as CO and, additionally, CO2.

[0239] The Wood-Ljungdahl pathway catalyzes the conversion of CO and
H2 to acetyl-CoA and other products such as acetate. Organisms
capable of utilizing CO and syngas also generally have the capability of
utilizing CO2 and CO2/H2 mixtures through the same basic
set of enzymes and transformations encompassed by the Wood-Ljungdahl
pathway. H2-dependent conversion of CO2 to acetate by
microorganisms was recognized long before it was revealed that CO also
could be used by the same organisms and that the same pathways were
involved. Many acetogens have been shown to grow in the presence of
CO2 and produce compounds such as acetate as long as hydrogen is
present to supply the necessary reducing equivalents (see for example,
Drake, Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:

2CO2+4H2+nADP+nPi→CH3COOH+2H2O+nATP

[0240] Hence, non-naturally occurring microorganisms possessing the
Wood-Ljungdahl pathway can utilize CO2 and H2 mixtures as well
for the production of acetyl-CoA and other desired products.

[0241] The Wood-Ljungdahl pathway is well known in the art and consists of
12 reactions which can be separated into two branches: (1) methyl branch
and (2) carbonyl branch. The methyl branch converts syngas to
methyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branch converts
methyl-THF to acetyl-CoA. The reactions in the methyl branch are
catalyzed in order by the following enzymes: ferredoxin oxidoreductase,
formate dehydrogenase, formyltetrahydrofolate synthetase,
methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate
dehydrogenase and methylenetetrahydrofolate reductase. The reactions in
the carbonyl branch are catalyzed in order by the following enzymes or
proteins: cobalamide corrinoid/iron-sulfur protein, methyltransferase,
carbon monoxide dehydrogenase, acetyl-CoA synthase, acetyl-CoA synthase
disulfide reductase and hydrogenase, and these enzymes can also be
referred to as methyltetrahydrofolate:corrinoid protein methyltransferase
(for example, AcsE), corrinoid iron-sulfur protein, nickel-protein
assembly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase,
carbon monoxide dehydrogenase and nickel-protein assembly protein (for
example, CooC). Following the teachings and guidance provided herein for
introducing a sufficient number of encoding nucleic acids to generate a
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
pathway, those skilled in the art will understand that the same
engineering design also can be performed with respect to introducing at
least the nucleic acids encoding the Wood-Ljungdahl enzymes or proteins
absent in the host organism. Therefore, introduction of one or more
encoding nucleic acids into the microbial organisms of the invention such
that the modified organism contains the complete Wood-Ljungdahl pathway
will confer syngas utilization ability.

[0242] Additionally, the reductive (reverse) tricarboxylic acid cycle
coupled with carbon monoxide dehydrogenase and/or hydrogenase activities
can also be used for the conversion of CO, CO2 and/or H2 to
acetyl-CoA and other products such as acetate. Organisms capable of
fixing carbon via the reductive TCA pathway can utilize one or more of
the following enzymes: ATP citrate-lyase, citrate lyase, aconitase,
isocitrate dehydrogenase, alpha-ketoglutarate:ferredoxin oxidoreductase,
succinyl-CoA synthetase, succinyl-CoA transferase, fumarate reductase,
fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon
monoxide dehydrogenase, and hydrogenase. Specifically, the reducing
equivalents extracted from CO and/or H2 by carbon monoxide
dehydrogenase and hydrogenase are utilized to fix CO2 via the
reductive TCA cycle into acetyl-CoA or acetate. Acetate can be converted
to acetyl-CoA by enzymes such as acetyl-CoA transferase, acetate
kinase/phosphotransacetylase, and acetyl-CoA synthetase. Acetyl-CoA can
be converted to the p-toluate, terepathalate, or
(2-hydroxy-3-methyl-4-oxobutoxy)phosphonate precursors,
glyceraldehyde-3-phosphate, phosphoenolpyruvate, and pyruvate, by
pyruvate:ferredoxin oxidoreductase and the enzymes of gluconeogenesis.
Following the teachings and guidance provided herein for introducing a
sufficient number of encoding nucleic acids to generate a p-toluate,
terephthalate or (2-hydroxy-3-methyl-4-oxobutoxy)phosphonate pathway,
those skilled in the art will understand that the same engineering design
also can be performed with respect to introducing at least the nucleic
acids encoding the reductive TCA pathway enzymes or proteins absent in
the host organism. Therefore, introduction of one or more encoding
nucleic acids into the microbial organisms of the invention such that the
modified organism contains the complete reductive TCA pathway will confer
syngas utilization ability.

[0243] Given the teachings and guidance provided herein, those skilled in
the art will understand that a non-naturally occurring microbial organism
can be produced that secretes the biosynthesized compounds of the
invention when grown on a carbon source such as a carbohydrate. Such
compounds include, for example, 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid and any of the intermediate
metabolites in the 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid pathway. All that is required is to engineer in one or
more of the required enzyme activities to achieve biosynthesis of the
desired compound or intermediate including, for example, inclusion of
some or all of the 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid biosynthetic pathways. Accordingly, the invention
provides a non-naturally occurring microbial organism that produces
and/or secretes 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid when grown on a carbohydrate and produces and/or secretes
any of the intermediate metabolites shown in the 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid pathway when grown on
a carbohydrate. For example, an adipate producing microbial organisms can
initiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA,
3-hydroxyadipyl-CoA, 5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see FIG.
2), as desired. In addition, an adipate producing microbial organism can
initiate synthesis from an intermediate, for example, 3-oxoadipyl-CoA,
3-oxoadipate, 3-hydroxyadipate, or hexa-2-enedioate (see FIG. 3). The
6-aminocaproic acid producing microbial organism of the invention can
initiate synthesis from an intermediate, for example, adipate
semialdehyde (see FIG. 8). The caprolactam producing microbial organism
of the invention can initiate synthesis from an intermediate, for
example, adipate semialdehyde or 6-aminocaproic acid (see FIG. 8), as
desired.

[0244] The non-naturally occurring microbial organisms of the invention
are constructed using methods well known in the art as exemplified herein
to exogenously express at least one nucleic acid encoding a
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
pathway enzyme in sufficient amounts to produce 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid. It is understood
that the microbial organisms of the invention are cultured under
conditions sufficient to produce 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid. Following the teachings and
guidance provided herein, the non-naturally occurring microbial organisms
of the invention can achieve biosynthesis of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid resulting in
intracellular concentrations between about 0.1-200 mM or more. Generally,
the intracellular concentration of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid is between about 3-150 mM,
particularly between about 5-125 mM and more particularly between about
8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more.
Intracellular concentrations between and above each of these exemplary
ranges also can be achieved from the non-naturally occurring microbial
organisms of the invention.

[0245] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well known in
the art. Exemplary anaerobic conditions for fermentation processes are
described herein and are described, for example, in U.S. publication
2009/0047719, filed Aug. 10, 2007. Any of these conditions can be
employed with the non-naturally occurring microbial organisms as well as
other anaerobic conditions well known in the art. Under such anaerobic
conditions, the 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid producers can synthesize 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid at intracellular concentrations of
5-10 mM or more as well as all other concentrations exemplified herein.
It is understood that, even though the above description refers to
intracellular concentrations, 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid producing microbial organisms can
produce 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid intracellularly and/or secrete the product into the
culture medium.

[0246] The culture conditions can include, for example, liquid culture
procedures as well as fermentation and other large scale culture
procedures. As described herein, particularly useful yields of the
biosynthetic products of the invention can be obtained under anaerobic or
substantially anaerobic culture conditions.

[0247] As described herein, one exemplary growth condition for achieving
biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid includes anaerobic culture or fermentation conditions. In
certain embodiments, the non-naturally occurring microbial organisms of
the invention can be sustained, cultured or fermented under anaerobic or
substantially anaerobic conditions. Briefly, anaerobic conditions refers
to an environment devoid of oxygen. Substantially anaerobic conditions
include, for example, a culture, batch fermentation or continuous
fermentation such that the dissolved oxygen concentration in the medium
remains between 0 and 10% of saturation. Substantially anaerobic
conditions also includes growing or resting cells in liquid medium or on
solid agar inside a sealed chamber maintained with an atmosphere of less
than 1% oxygen. The percent of oxygen can be maintained by, for example,
sparging the culture with an N2/CO2 mixture or other suitable
non-oxygen gas or gases.

[0248] The culture conditions described herein can be scaled up and grown
continuously for manufacturing of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid. Exemplary growth procedures
include, for example, fed-batch fermentation and batch separation;
fed-batch fermentation and continuous separation, or continuous
fermentation and continuous separation. All of these processes are well
known in the art. Fermentation procedures are particularly useful for the
biosynthetic production of commercial quantities of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid. Generally, and as
with non-continuous culture procedures, the continuous and/or
near-continuous production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid will include culturing a
non-naturally occurring 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid producing organism of the
invention in sufficient nutrients and medium to sustain and/or nearly
sustain growth in an exponential phase. Continuous culture under such
conditions can include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or
more. Additionally, continuous culture can include 1 week, 2, 3, 4 or 5
or more weeks and up to several months. Alternatively, organisms of the
invention can be cultured for hours, if suitable for a particular
application. It is to be understood that the continuous and/or
near-continuous culture conditions also can include all time intervals in
between these exemplary periods. It is further understood that the time
of culturing the microbial organism of the invention is for a sufficient
period of time to produce a sufficient amount of product for a desired
purpose.

[0249] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid can be utilized in,
for example, fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation, or continuous fermentation and
continuous separation. Examples of batch and continuous fermentation
procedures are well known in the art.

[0250] In addition to the above fermentation procedures using the
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
producers of the invention for continuous production of substantial
quantities of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid, the 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid producers also can be, for
example, simultaneously subjected to chemical synthesis procedures to
convert the product to other compounds or the product can be separated
from the fermentation culture and sequentially subjected to chemical
conversion to convert the product to other compounds, if desired. As
described herein, an intermediate in the adipate pathway utilizing
3-oxoadipate, hexa-2-enedioate, can be converted to adipate, for example,
by chemical hydrogenation over a platinum catalyst (see Example III).

[0251] As described herein, exemplary growth conditions for achieving
biosynthesis of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid includes the addition of an osmoprotectant to the
culturing conditions. In certain embodiments, the non-naturally occurring
microbial organisms of the invention can be sustained, cultured or
fermented as described above in the presence of an osmoprotectant.
Briefly, an osmoprotectant means a compound that acts as an osmolyte and
helps a microbial organism as described herein survive osmotic stress.
Osmoprotectants include, but are not limited to, betaines, amino acids,
and the sugar trehalose. Non-limiting examples of such are glycine
betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one aspect,
the osmoprotectant is glycine betaine. It is understood to one of
ordinary skill in the art that the amount and type of osmoprotectant
suitable for protecting a microbial organism described herein from
osmotic stress will depend on the microbial organism used. For example,
as described in Example XXII, Escherichia coli in the presence of varying
amounts of 6-aminocaproic acid is suitably grown in the presence of 2 mM
glycine betaine. The amount of osmoprotectant in the culturing conditions
can be, for example, no more than about 0.1 mM, no more than about 0.5
mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than
about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no
more than about 5.0 mM, no more than about 7.0 mM, no more than about 10
mM, no more than about 50 mM, no more than about 100 mM or no more than
about 500 mM.

[0252] To generate better producers, metabolic modeling can be utilized to
optimize growth conditions. Modeling can also be used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid.

[0253] One computational method for identifying and designing metabolic
alterations favoring biosynthesis of a desired product is the OptKnock
computational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657
(2003). OptKnock is a metabolic modeling and simulation program that
suggests gene deletion strategies that result in genetically stable
microorganisms which overproduce the target product. Specifically, the
framework examines the complete metabolic and/or biochemical network of a
microorganism in order to suggest genetic manipulations that force the
desired biochemical to become an obligatory byproduct of cell growth. By
coupling biochemical production with cell growth through strategically
placed gene deletions or other functional gene disruption, the growth
selection pressures imposed on the engineered strains after long periods
of time in a bioreactor lead to improvements in performance as a result
of the compulsory growth-coupled biochemical production. Lastly, when
gene deletions are constructed there is a negligible possibility of the
designed strains reverting to their wild-type states because the genes
selected by OptKnock are to be completely removed from the genome.
Therefore, this computational methodology can be used to either identify
alternative pathways that lead to biosynthesis of a desired product or
used in connection with the non-naturally occurring microbial organisms
for further optimization of biosynthesis of a desired product.

[0254] The concept of growth-coupled biochemical production can be
visualized in the context of the biochemical production envelopes of a
typical metabolic network calculated using an in silico model. These
limits are obtained by fixing the uptake rate(s) of the limiting
substrate(s) to their experimentally measured value(s) and calculating
the maximum and minimum rates of biochemical production at each
attainable level of growth. Although exceptions exist, typically the
production of a desired biochemical is in direct competition with biomass
formation for intracellular resources. Thus, enhanced rates of
biochemical production will necessarily result in sub-maximal growth
rates. The knockouts suggested by OptKnock are designed to restrict the
allowable solution boundaries forcing a change in metabolic behavior from
the wild-type strain. Although the actual solution boundaries for a given
strain will expand or contract as the substrate uptake rate(s) increase
or decrease, each experimental point should lie within its calculated
solution boundary. Plots such as these allow one to visualize how close
strains are to their performance limits or, in other words, how much room
is available for improvement. The OptKnock framework has already been
able to identify promising gene deletion strategies for biochemical
overproduction, (Burgard et al., Biotechnol Bioeng, 84(6):647-657 (2003);
Pharkya et al., Biotechnol Bioeng, 84(7):887-899 (2003)) and establishes
a systematic framework that will naturally encompass future improvements
in metabolic and regulatory modeling frameworks.

[0255] Briefly, OptKnock is a term used herein to refer to a computational
method and system for modeling cellular metabolism. The OptKnock program
relates to a framework of models and methods that incorporate particular
constraints into flux balance analysis (FBA) models. These constraints
include, for example, qualitative kinetic information, qualitative
regulatory information, and/or DNA microarray experimental data. OptKnock
also computes solutions to various metabolic problems by, for example,
tightening the flux boundaries derived through flux balance models and
subsequently probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational framework
allows the construction of model formulations that enable an effective
query of the performance limits of metabolic networks and provides
methods for solving the resulting mixed-integer linear programming
problems. The metabolic modeling and simulation method referred to herein
as OptKnock are described in, for example, U.S. publication 2002/0168654,
filed Jan. 10, 2002, in International Patent No. PCT/US02/00660, filed
Jan. 10, 2002, and U.S. patent application serial No. 2009/0047719, filed
Aug. 10, 2007.

[0256] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product is a
metabolic modeling and simulation system termed SimPheny®. This
computational method and system is described in, for example, U.S.
publication 2003/0233218, filed Jun. 14, 2002, and in International
Patent Application No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny®
is a computational system that can be used to produce a network model in
silico and to simulate the flux of mass, energy or charge through the
chemical reactions of a biological system to define a solution space that
contains any and all possible functionalities of the chemical reactions
in the system, thereby determining a range of allowed activities for the
biological system. This approach is referred to as constraints-based
modeling because the solution space is defined by constraints such as the
known stoichiometry of the included reactions as well as reaction
thermodynamic and capacity constraints associated with maximum fluxes
through reactions. The space defined by these constraints can be
interrogated to determine the phenotypic capabilities and behavior of the
biological system or of its biochemical components. Analysis methods such
as convex analysis, linear programming and the calculation of extreme
pathways as described, for example, in Schilling et al., J. Theon. Biol.
203:229-248 (2000); Schilling et al., Biotech. Bioeng. 71:286-306 (2000)
and Schilling et al., Biotech. Prog. 15:288-295 (1999), can be used to
determine such phenotypic capabilities.

[0257] As described above, one constraints-based method used in the
computational programs applicable to the invention is flux balance
analysis. Flux balance analysis is based on flux balancing in a steady
state condition and can be performed as described in, for example, Varma
and Palsson, Biotech. Bioeng. 12:994-998 (1994). Flux balance approaches
have been applied to reaction networks to simulate or predict systemic
properties of, for example, adipocyte metabolism as described in Fell and
Small, J. Biochem. 138:781-786 (1986), acetate secretion from E. coli
under ATP maximization conditions as described in Majewski and Domach,
Biotech. Bioeng. 35:732-738 (1990) or ethanol secretion by yeast as
described in Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996).
Additionally, this approach can be used to predict or simulate the growth
of S. cerevisiae on a variety of single-carbon sources as well as the
metabolism of H. influenzae as described in Edwards and Palsson, Proc.
Natl. Acad. Sci. 97:5528-5533 (2000), Edwards and Palsson, J. Bio. Chem.
274:17410-17416 (1999) and Edwards et al., Nature Biotech. 19:125-130
(2001).

[0258] Once the solution space has been defined, it can be analyzed to
determine possible solutions under various conditions. This computational
approach is consistent with biological realities because biological
systems are flexible and can reach the same result in many different
ways. Biological systems are designed through evolutionary mechanisms
that have been restricted by fundamental constraints that all living
systems must face. Therefore, constraints-based modeling strategy
embraces these general realities. Further, the ability to continuously
impose further restrictions on a network model via the tightening of
constraints results in a reduction in the size of the solution space,
thereby enhancing the precision with which physiological performance or
phenotype can be predicted.

[0259] These computational approaches are consistent with biological
realities because biological systems are flexible and can reach the same
result in many different ways. Biological systems are designed through
evolutionary mechanisms that have been restricted by fundamental
constraints that all living systems must face. Therefore,
constraints-based modeling strategy embraces these general realities.
Further, the ability to continuously impose further restrictions on a
network model via the tightening of constraints results in a reduction in
the size of the solution space, thereby enhancing the precision with
which physiological performance or phenotype can be predicted.

[0260] Given the teachings and guidance provided herein, those skilled in
the art will be able to apply various computational frameworks for
metabolic modeling and simulation to design and implement biosynthesis of
a desired compound in host microbial organisms. Such metabolic modeling
and simulation methods include, for example, the computational systems
exemplified above as SimPheny® and OptKnock. For illustration of the
invention, some methods are described herein with reference to the
OptKnock computation framework for modeling and simulation. Those skilled
in the art will know how to apply the identification, design and
implementation of the metabolic alterations using OptKnock to any of such
other metabolic modeling and simulation computational frameworks and
methods well known in the art.

[0261] The ability of a cell or organism to obligatory couple growth to
the production of a biochemical product can be illustrated in the context
of the biochemical production limits of a typical metabolic network
calculated using an in silico model. These limits are obtained by fixing
the uptake rate(s) of the limiting substrate(s) to their experimentally
measured value(s) and calculating the maximum and minimum rates of
biochemical production at each attainable level of growth. The production
of a desired biochemical generally is in direct competition with biomass
formation for intracellular resources. Under these circumstances,
enhanced rates of biochemical production will necessarily result in
sub-maximal growth rates. The knockouts suggested by the above metabolic
modeling and simulation programs such as OptKnock are designed to
restrict the allowable solution boundaries forcing a change in metabolic
behavior from the wild-type strain. Although the actual solution
boundaries for a given strain will expand or contract as the substrate
uptake rate(s) increase or decrease, each experimental point will lie
within its calculated solution boundary. Plots such as these allow
accurate predictions of how close the designed strains are to their
performance limits which also indicates how much room is available for
improvement.

[0262] The OptKnock mathematical framework is exemplified herein for
pinpointing gene deletions leading to growth-coupled biochemical
production (see Example XXX). The procedure builds upon constraint-based
metabolic modeling which narrows the range of possible phenotypes that a
cellular system can display through the successive imposition of
governing physico-chemical constraints, Price et al., Nat Rev Microbiol,
2: 886-97 (2004). As described above, constraint-based models and
simulations are well known in the art and generally invoke the
optimization of a particular cellular objective, subject to network
stoichiometry, to suggest a likely flux distribution.

[0263] Briefly, the maximization of a cellular objective quantified as an
aggregate reaction flux for a steady state metabolic network comprising a
set N={1, . . . , N} of metabolites and a set M={1, . . . , M} of
metabolic reactions is expressed mathematically as follows:

where Sij is the stoichiometric coefficient of metabolite i in
reaction j, vj is the flux of reaction j,
vsubstrate--uptake represents the assumed or measured uptake
rate(s) of the limiting substrate(s), and vatp--main is the
non-growth associated ATP maintenance requirement. The vector v includes
both internal and external fluxes. In this study, the cellular objective
is often assumed to be a drain of biosynthetic precursors in the ratios
required for biomass formation, Neidhardt, F. C. et al., 2nd ed. 1996,
Washington, D.C.: ASM Press. 2 v. (xx, 2822, 1xxvi). The fluxes are
generally reported per 1 gDWhr (gram of dry weight times hour) such that
biomass formation is expressed as g biomass produced/gDWhr or 1/hr.

assume a value of 11f reaction j is active and a value of 0 if it is
inactive. The following constraint,

vjminyj≦vj≦vjmaxyj,.A-i-
nverted.jεM

ensures that reaction flux vj is set to zero only if variable
yj is equal to zero. Alternatively, when yj is equal to one,
vj is free to assume any value between a lower vjmin and
an upper vjmax bound. Here, vjmin and vjmax
are identified by minimizing and maximizing, respectively, every reaction
flux subject to the network constraints described above, Mahadevan et
al., Metab Eng, 5: 264-76 (2003).

[0265] Optimal gene/reaction knockouts are identified by solving a bilevel
optimization problem that chooses the set of active reactions (yj=1)
such that an optimal growth solution for the resulting network
overproduces the chemical of interest. Schematically, this bilevel
optimization problem is illustrated in FIG. 2. Mathematically, this
bilevel optimization problem is expressed as the following bilevel
mixed-integer optimization problem:

where vchemical is the production of the desired target product, for
example adipate, 6-ACA and/or HMDA, or other biochemical product, and K
is the number of allowable knockouts. Note that setting K equal to zero
returns the maximum biomass solution of the complete network, while
setting K equal to one identifies the single gene/reaction knockout
(yj=0) such that the resulting network involves the maximum
overproduction given its maximum biomass yield. The final constraint
ensures that the resulting network meets a minimum biomass yield. Burgard
et al., Biotechnol Bioeng, 84: 647-57 (2003), provide a more detailed
description of the model formulation and solution procedure. Problems
containing hundreds of binary variables can be solved in the order of
minutes to hours using CPLEX 8.0, GAMS: The Solver Manuals. 2003: GAMS
Development Corporation, accessed via the GAMS, Brooke et al., GAMS
Development Corporation (1998), modeling environment on an IBM RS6000-270
workstation. The OptKnock framework has already been able to identify
promising gene deletion strategies for biochemical overproduction,
Burgard et al., Biotechnol Bioeng, 84: 647-57 (2003), Pharkya et al.,
Biotechnol Bioeng, 84: 887-899 (2003), and establishes a systematic
framework that will naturally encompass future improvements in metabolic
and regulatory modeling frameworks.

[0266] The methods described above will provide one set of metabolic
reactions to disrupt. Elimination of each reaction within the set or
metabolic modification can result in a desired product as an obligatory
product during the growth phase of the organism. Because the reactions
are known, a solution to the bilevel OptKnock problem also will provide
the associated gene or genes encoding one or more enzymes that catalyze
each reaction within the set of reactions. Identification of a set of
reactions and their corresponding genes encoding the enzymes
participating in each reaction is generally an automated process,
accomplished through correlation of the reactions with a reaction
database having a relationship between enzymes and encoding genes.

[0267] Once identified, the set of reactions that are to be disrupted in
order to achieve production of a desired product are implemented in the
target cell or organism by functional disruption of at least one gene
encoding each metabolic reaction within the set. One particularly useful
means to achieve functional disruption of the reaction set is by deletion
of each encoding gene. However, in some instances, it can be beneficial
to disrupt the reaction by other genetic aberrations including, for
example, mutation, deletion of regulatory regions such as promoters or
cis binding sites for regulatory factors, or by truncation of the coding
sequence at any of a number of locations. These latter aberrations,
resulting in less than total deletion of the gene set can be useful, for
example, when rapid assessments of the coupling of a product are desired
or when genetic reversion is less likely to occur.

[0268] To identify additional productive solutions to the above described
bilevel OptKnock problem which lead to further sets of reactions to
disrupt or metabolic modifications that can result in the biosynthesis,
including growth-coupled biosynthesis of a desired product, an
optimization method, termed integer cuts, can be implemented. This method
proceeds by iteratively solving the OptKnock problem exemplified above
with the incorporation of an additional constraint referred to as an
integer cut at each iteration. Integer cut constraints effectively
prevent the solution procedure from choosing the exact same set of
reactions identified in any previous iteration that obligatorily couples
product biosynthesis to growth. For example, if a previously identified
growth-coupled metabolic modification specifies reactions 1, 2, and 3 for
disruption, then the following constraint prevents the same reactions
from being simultaneously considered in subsequent solutions. The integer
cut method is well known in the art and can be found described in, for
example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all
methods described herein with reference to their use in combination with
the OptKnock computational framework for metabolic modeling and
simulation, the integer cut method of reducing redundancy in iterative
computational analysis also can be applied with other computational
frameworks well known in the art including, for example, SimPheny®.

[0269] The methods exemplified herein allow the construction of cells and
organisms that biosynthetically produce a desired product, including the
obligatory coupling of production of a target biochemical product to
growth of the cell or organism engineered to harbor the identified
genetic alterations. Therefore, the computational methods described
herein allow the identification and implementation of metabolic
modifications that are identified by an in silico method selected from
OptKnock or SimPheny®. The set of metabolic modifications can
include, for example, addition of one or more biosynthetic pathway
enzymes and/or functional disruption of one or more metabolic reactions
including, for example, disruption by gene deletion.

[0270] As discussed above, the OptKnock methodology was developed on the
premise that mutant microbial networks can be evolved towards their
computationally predicted maximum-growth phenotypes when subjected to
long periods of growth selection. In other words, the approach leverages
an organism's ability to self-optimize under selective pressures. The
OptKnock framework allows for the exhaustive enumeration of gene deletion
combinations that force a coupling between biochemical production and
cell growth based on network stoichiometry. The identification of optimal
gene/reaction knockouts requires the solution of a bilevel optimization
problem that chooses the set of active reactions such that an optimal
growth solution for the resulting network overproduces the biochemical of
interest (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)).

[0271] An in silico stoichiometric model of E. coli metabolism can be
employed to identify essential genes for metabolic pathways as
exemplified previously and described in, for example, U.S. patent
publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US
2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and
in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock
mathematical framework can be applied to pinpoint gene deletions leading
to the growth-coupled production of a desired product. Further, the
solution of the bilevel OptKnock problem provides only one set of
deletions. To enumerate all meaningful solutions, that is, all sets of
knockouts leading to growth-coupled production formation, an optimization
technique, termed integer cuts, can be implemented. This entails
iteratively solving the OptKnock problem with the incorporation of an
additional constraint referred to as an integer cut at each iteration, as
discussed above.

[0272] Given the teachings and guidance provided herein, those skilled in
the art will understand that to disrupt an enzymatic reaction the
catalytic activity of the one or more enzymes involved in the reaction is
to be disrupted. Disruption can occur by a variety of means including,
for example, deletion of an encoding gene or incorporation of a genetic
alteration in one or more of the encoding gene sequences. The encoding
genes targeted for disruption can be one, some, or all of the genes
encoding enzymes involved in the catalytic activity. For example, where a
single enzyme is involved in a targeted catalytic activity disruption can
occur by a genetic alteration that reduces or destroys the catalytic
activity of the encoded gene product. Similarly, where the single enzyme
is multimeric, including heteromeric, disruption can occur by a genetic
alteration that reduces or destroys the function of one or all subunits
of the encoded gene products. Destruction of activity can be accomplished
by loss of the binding activity of one or more subunits in order to form
an active complex, by destruction of the catalytic subunit of the
multimeric complex or by both. Other functions of multimeric protein
association and activity also can be targeted in order to disrupt a
metabolic reaction of the invention. Such other functions are well known
to those skilled in the art. Further, some or all of the functions of a
single polypeptide or multimeric complex can be disrupted according to
the invention in order to reduce or abolish the catalytic activity of one
or more enzymes involved in a reaction or metabolic modification of the
invention. Similarly, some or all of enzymes involved in a reaction or
metabolic modification of the invention can be disrupted so long as the
targeted reaction is reduced or eliminated.

[0273] Given the teachings and guidance provided herein, those skilled in
the art also will understand that an enzymatic reaction can be disrupted
by reducing or eliminating reactions encoded by a common gene and/or by
one or more orthologs of that gene exhibiting similar or substantially
the same activity. Reduction of both the common gene and all orthologs
can lead to complete abolishment of any catalytic activity of a targeted
reaction. However, disruption of either the common gene or one or more
orthologs can lead to a reduction in the catalytic activity of the
targeted reaction sufficient to promote coupling of growth to product
biosynthesis. Exemplified herein are both the common genes encoding
catalytic activities for a variety of metabolic modifications as well as
their orthologs. Those skilled in the art will understand that disruption
of some or all of the genes encoding a enzyme of a targeted metabolic
reaction can be practiced in the methods of the invention and
incorporated into the non-naturally occurring microbial organisms of the
invention in order to achieve the growth-coupled product production.
Examplary disruptions to confer increased production of adipate, 6-ACA
and/or HMDA are described in Example XXX and Tables 14-16.

[0274] Employing the methods exemplified above, the methods of the
invention allow the construction of cells and organisms that increase
production of a desired product, for example, by coupling the production
of a desired product to growth of the cell or organism engineered to
harbor the identified genetic alterations. As disclosed herein, metabolic
alterations have been identified that couple the production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
to growth of the organism. Microbial organism strains constructed with
the identified metabolic alterations produce elevated levels, relative to
the absence of the metabolic alterations, of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid during the
exponential growth phase. These strains can be beneficially used for the
commercial production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid in continuous fermentation process
without being subjected to the negative selective pressures described
previously. Although exemplified herein as metabolic alterations, in
particular one or more gene disruptions, that confer growth coupled
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid, it is understood that any gene disruption that increases
the production of 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid can be introduced into a host microbial organism, as
desired.

[0275] Therefore, the methods of the invention provide a set of metabolic
modifications that are identified by an in silico method such as
OptKnock. The set of metabolic modifications can include functional
disruption of one or more metabolic reactions including, for example,
disruption by gene deletion. For 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid production, metabolic
modifications can be selected from the set of metabolic modifications
listed in Tables 14-16 (see Example XXX).

[0276] Also provided is a method of producing a non-naturally occurring
microbial organisms having stable growth-coupled production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid.
The method can include identifying in silico a set of metabolic
modifications that increase production of 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid, for example,
increase production during exponential growth; genetically modifying an
organism to contain the set of metabolic modifications that increase
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid, and culturing the genetically modified organism. If
desired, culturing can include adaptively evolving the genetically
modified organism under conditions requiring production of 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid. The methods of
the invention are applicable to bacterium, yeast and fungus as well as a
variety of other cells and microorganism, as disclosed herein.

[0277] Thus, the invention provides a non-naturally occurring microbial
organism comprising one or more gene disruptions that confer increased
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid. In one embodiment, the one or more gene disruptions
confer growth-coupled production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid, and can, for example, confer
stable growth-coupled production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid. In another embodiment, the one or
more gene disruptions can confer obligatory coupling of 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid production to
growth of the microbial organism. Such one or more gene disruptions
reduce the activity of the respective one or more encoded enzymes.

[0278] The non-naturally occurring microbial organism can have one or more
gene disruptions included in a metabolic modification listed in Tables
14-16. As disclosed herein, the one or more gene disruptions can be a
deletion. Such non-naturally occurring microbial organisms of the
invention include bacteria, yeast, fungus, or any of a variety of other
microorganisms applicable to fermentation processes, as disclosed herein.

[0279] Thus, the invention provides a non-naturally occurring microbial
organism, comprising one or more gene disruptions, where the one or more
gene disruptions occur in genes encoding proteins or enzymes where the
one or more gene disruptions confer increased production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
in the organism. The production of 6-aminocaproic acid, caprolactam,
hexamethylenediamine or levulinic acid can be growth-coupled or not
growth-coupled. In a particular embodiment, the production of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
can be obligatorily coupled to growth of the organism, as disclosed
herein.

[0280] The invention provides non naturally occurring microbial organisms
having genetic alterations such as gene disruptions that increase
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid, for example, growth-coupled production of 6-aminocaproic
acid, caprolactam, hexamethylenediamine or levulinic acid. Product
production can be, for example, obligatorily linked to the exponential
growth phase of the microorganism by genetically altering the metabolic
pathways of the cell, as disclosed herein. The genetic alterations can
increase the production of the desired product or even make the desired
product an obligatory product during the growth phase. Sets of metabolic
alterations or transformations that result in increased production and
elevated levels of 6-aminocaproic acid, caprolactam, hexamethylenediamine
or levulinic acid biosynthesis are exemplified in Tables 14-16 (see
Example XXX). Each alteration within a set corresponds to the requisite
metabolic reaction that should be functionally disrupted. Functional
disruption of all reactions within each set can result in the increased
production of 6-aminocaproic acid, caprolactam, hexamethylenediamine or
levulinic acid by the engineered strain during the growth phase. The
corresponding reactions to the referenced alterations can be found in
Tables 14-16 (see Example XXX), and the gene or genes that encode enzymes
or proteins that carry out the reactions are set forth in Tables 14-16.

[0281] For example, for each strain exemplified in Tables 14-16, the
metabolic alterations that can be generated for 6-aminocaproic acid,
caprolactam, hexamethylenediamine or levulinic acid production are shown
in each row. These alterations include the functional disruption of the
reactions shown in Tables 14-16. Each of these non-naturally occurring
alterations result in increased production and an enhanced level of
6-aminocaproic acid, caprolactam, hexamethylenediamine or levulinic acid
production, for example, during the exponential growth phase of the
microbial organism, compared to a strain that does not contain such
metabolic alterations, under appropriate culture conditions. Appropriate
conditions include, for example, those disclosed herein, including
conditions such as particular carbon sources or reactant availabilities
and/or adaptive evolution.

[0282] It is understood that modifications which do not substantially
affect the activity of the various embodiments of this invention are also
provided within the definition of the invention provided herein.
Accordingly, the following examples are intended to illustrate but not
limit the present invention.

[0284] Organisms such as Penicillium chrysogenum have the ability to
naturally degrade adipate (Thykaer et al., Metab. Eng. 4:151-158.
(2002)). The mechanism is similar to the oxidation of fatty acids (see
FIG. 1). The first step in adipate degradation is an ATP-dependent
reaction that activates adipate with CoA. The second reaction is
catalyzed by a dehydrogenase that forms 5-carboxy-2-pentenoyl-CoA from
adipyl-CoA. During peroxisomal adipate degradation, the dehydrogenase
enzyme contains FAD, which accepts the electrons and then transfers them
directly to oxygen. A catalase enzyme dissipates the H2O2
formed by the reduction of oxygen. In mitochondrial fatty acid oxidation,
the FAD from the dehydrogenase transfers electrons directly to the
electron transport chain. A multi-functional fatty acid oxidation protein
in eukaryotes such as S. cerevisiae and P. chrysogenum carries out the
following hydratase and dehydrogenase steps. The final step is an acyl
transferase that splits 3-oxoadipyl CoA into acetyl-CoA and succinyl-CoA.

[0285] A highly efficient pathway for the production of adipate is
achieved through genetically altering a microorganism such that similar
enzymatic reactions are employed for adipate synthesis from succinyl-CoA
and acetyl-CoA (see FIG. 2). Successful implementation of this entails
expressing the appropriate genes, tailoring their expression, and
altering culture conditions so that high acetyl-CoA, succinyl-CoA, and/or
redox (for example, NADH/NAD+) ratios will drive the metabolic flux
through this pathway in the direction of adipate synthesis rather than
degradation. Strong parallels to butyrate formation in Clostridia
(Kanehisa and Goto, Nucl. Acids Res. 28:27-30 (2000)) support that each
step in the adipate synthesis pathway is thermodynamically feasible with
reaction directionality governed by the concentrations of the
participating metabolites. The final step, which forms adipate from
adipyl-CoA, can take place either via a synthetase,
phosphotransadipylase/kinase, transferase, or hydrolase mechanism.

[0286] The maximum theoretical yields of adipate using this pathway were
calculated both in the presence and absence of an external electron
acceptor such as oxygen. These calculations show that the pathway can
efficiently transform glucose into adipate and CO2 under anaerobic
conditions with a 92% molar yield (Table 1). The production of adipate
using this pathway does not require the uptake of oxygen as NAD+ can be
regenerated in the two hydrogenase steps that form 3-hydroxyadipyl-CoA
and adipyl-CoA (see FIG. 2). Further, the pathway is favorable
energetically as up to 1.55 moles of ATP are formed per mole of glucose
consumed at the maximum theoretical yield of adipate assuming either a
synthetase, phosphotransadipylase/kinase, or transferase mechanism for
the final conversion step. The ATP yield can be further improved to 2.47
moles of ATP produced per mole of glucose if phosphoenolpyruvate
carboxykinase (PPCK) is assumed to function in the ATP-generating
direction towards oxaloacetate formation. Maximum ATP yield calculations
were then performed assuming that the adipyl-CoA to adipate
transformation is a hydrolysis step. This reduces the maximum ATP yields
at maximum adipate production to 0.85 and 1.77 mole ATP per mole glucose
consumed if PPCK is assumed irreversible and reversible, respectively.
Nevertheless, these ATP yields are sufficient for cell growth,
maintenance, and production.

[0287] Successfully engineering this pathway involves identifying an
appropriate set of enzymes with sufficient activity and specificity. This
entails identifying an appropriate set of enzymes, cloning their
corresponding genes into a production host, optimizing fermentation
conditions, and assaying for product formation following fermentation. To
engineer a production host for the production of adipate, one or more
exogenous DNA sequence(s) are expressed in a suitable host microorganism.
In addition, the microorganisms can have endogenous gene(s) functionally
deleted. These modifications allow the production of adipate using
renewable feedstock.

[0288] Below is described a number of biochemically characterized
candidate genes that encode enzymes that catalyze each step of the
reverse adipate degradation pathway in a production host. Although
described using E. coli as a host organism to engineer the pathway,
essentially any suitable host organism can be used. Specifically listed
are genes that are native to E. coli as well as genes in other organisms
that can be applied to catalyze the appropriate transformations when
properly cloned and expressed.

[0289] Referring to FIG. 2, step I involves succinyl CoA:acetyl CoA acyl
transferase (β-ketothiolase). The first step in the pathway combines
acetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. The gene products
encoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J.
Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera et
al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), paaE in Pseudomonas
fluorescens ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)),
and paaJ from E. coli (Nogales et al., Microbiol. 153:357-365 (2007))
catalyze the conversion of 3-oxoadipyl-CoA into succinyl-CoA and
acetyl-CoA during the degradation of aromatic compounds such as
phenylacetate or styrene. Since β-ketothiolase enzymes catalyze
reversible transformations, these enzymes can be employed for the first
step in adipate synthesis shown in FIG. 2. For example, the ketothiolase
phaA from R. eutropha combines two molecules of acetyl-CoA to form
acetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)).
Similarly, a β-keto thiolase (bktB) has been reported to catalyze
the condensation of acetyl-CoA and propionyl-CoA to form
β-ketovaleryl-CoA (Slater et al., J. Bacteriol. 180: 1979-1987
(1998)) in R. eutropha. Additional candidates are found in Burkholderia
ambifaria AMMD. The protein sequences for the above-mentioned gene
products are well known in the art and can be accessed in the public
databases such as GenBank using the following GI numbers and/or GenBank
identifiers:

[0290] These exemplary sequences can be used to identify homologue
proteins in GenBank or other databases through sequence similarity
searches (for example, BLASTp). The resulting homologue proteins and
their corresponding gene sequences provide additional exogenous DNA
sequences for transformation into E. coli or other suitable host
microorganisms to generate production hosts.

[0291] For example, orthologs of paaJ from Escherichia coli K12 can be
found using the following GI numbers and/or GenBank identifiers:

[0294] It is less desirable to use the thiolase-encoding genes fadA and
fadB, genes in fatty acid degradation pathway in E. coli, in this
exemplary pathway. These genes form a complex that encodes for multiple
activities, most of which are not desired in this pathway.

[0295] Referring to FIG. 2, step 2 involves 3-hydroxyacyl-CoA
dehydrogenase. The second step in the pathway involves the reduction of
3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. The gene products encoded by phaC
in Pseudomonas putida U (Olivera et al., Proc. Natl. Acad. Sci. USA
95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro
et al., Arch. Microbiol. 188:117-125 (2007)) catalyze the reverse
reaction, that is, the oxidation of 3-hydroxyadipyl-CoA to form
3-oxoadipyl-CoA, during the catabolism of phenylacetate or styrene. The
reactions catalyzed by such dehydrogenases are reversible and accordingly
these genes represent candidates to carry out the second step of adipate
synthesis as shown in FIG. 2. A similar transformation is also carried
out by the gene product of hbd in Clostridium acetobutylicum (Atsumi et
al., Metab. Eng. (epub Sep. 14, 2007); Boynton et al., J. Bacteriol.
178:3015-3024 (1996)). This enzyme converts acetoacetyl-CoA to
3-hydroxybutyryl-CoA. Lastly, given the proximity in E. coli of paaH to
other genes in the phenylacetate degradation operon (Nogales et al.,
Microbiol. 153:357-365 (2007)) and the fact that paaH mutants cannot grow
on phenylacetate (Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)),
it is expected that the E. coli paaH gene encodes a 3-hydroxyacyl-CoA
dehydrogenase. The protein sequences for each of these exemplary gene
products can be found using the following GI numbers and/or GenBank
identifiers:

[0297] Alternatively, beta-oxidation genes are candidates for the first
three steps in adipate synthesis. Candidate genes for the proposed
adipate synthesis pathway also include the native fatty acid oxidation
genes of E. coli and their homologs in other organisms. The E. coli genes
fadA and fadB encode a multienzyme complex that exhibits ketoacyl-CoA
thiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase
activities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al., J.
Biol. Chem. 265:10424-10429 (1990); Yang et al., J. Biol. Chem. 266:16255
(1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937 (1990)).
These activities are mechanistically similar to the first three
transformations shown in FIG. 2. The fadI andfadJgenes encode similar
functions and are naturally expressed only anaerobically (Campbell et
al., Mol. Microbiol. 47:793-805 (2003)). These gene products naturally
operate to degrade short, medium, and long chain fatty-acyl-CoA compounds
to acetyl-CoA, rather than to convert succinyl-CoA and acetyl-CoA into
5-carboxy-2-pentenoyl-CoA as proposed in FIG. 2. However, it is well
known that the ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase,
and enoyl-CoA hydratase enzymes catalyze reversible transformations.
Furthermore, directed evolution and related approaches can be applied to
tailor the substrate specificities of the native beta-oxidation machinery
of E. coli. Thus these enzymes or homologues thereof can be applied for
adipate production. If the native genes operate to degrade adipate or its
precursors in vivo, the appropriate genetic modifications are made to
attenuate or eliminate these functions. However, it may not be necessary
since a method for producing poly[(R)-3-hydroxybutyrate] in E. coli that
involves activating fadB, by knocking out a negative regulator, fadR, and
co-expressing a non-native ketothiolase, phaA from Ralstonia eutropha,
has been described (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)).
This work clearly demonstrated that a beta-oxidation enzyme, in
particular the gene product of fadB which encodes both 3-hydroxyacyl-CoA
dehydrogenase and enoyl-CoA hydratase activities, can function as part of
a pathway to produce longer chain molecules from acetyl-CoA precursors.
The protein sequences for each of these exemplary gene products can be
found using the following GI numbers and/or GenBank identifiers:

[0299] One candidate gene for the enoyl-CoA reductase step is the gene
product of bcd from C. acetobutylicum (Atsumi et al., supra, 2007;
Boynton et al., J. Bacteriol. 178:3015-3024 (1996)), which naturally
catalyzes the reduction of crotonyl-CoA to butyryl-CoA, a reaction
similar in mechanism to the desired reduction of
5-carboxy-2-pentenoyl-CoA to adipyl-CoA in the adipate synthesis pathway.
Activity of this enzyme can be enhanced by expressing bcd in conjunction
with expression of the C. acetobutylicum etfAB genes, which encode an
electron transfer flavoprotein. An additional candidate for the enoyl-CoA
reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis
(Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct
derived from this sequence following the removal of its mitochondrial
targeting leader sequence was cloned in E. coli, resulting in an active
enzyme (Hoffmeister et al., supra, 2005). This approach is well known to
those skilled in the art of expressing eukarytotic genes, particularly
those with leader sequences that may target the gene product to a
specific intracellular compartment, in prokaryotic organisms. A close
homolog of this gene, TDE0597, from the prokaryote Treponema denticola
represents a third enoyl-CoA reductase which has been cloned and
expressed in E. coli (Tucci and Martin, FEBS Lett. 581:1561-1566 (2007)).
The protein sequences for each of these exemplary gene products can be
found using the following GI numbers and/or GenBank identifiers:

[0300] Referring to FIG. 2, step 5 involves adipyl-CoA synthetase (also
referred to as adipate-CoA ligase), phosphotransadipylase/adipate kinase,
adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. From an
energetic standpoint, it is desirable for the final step in the adipate
synthesis pathway to be catalyzed by an enzyme or enzyme pair that can
conserve the ATP equivalent stored in the thioester bond of adipyl-CoA.
The product of the sucC and sucD genes of E. coli, or homologs thereof,
can potentially catalyze the final transformation shown in FIG. 2 should
they exhibit activity on adipyl-CoA. The sucCD genes naturally form a
succinyl-CoA synthetase complex that catalyzes the formation of
succinyl-CoA from succinate with the concaminant consumption of one ATP,
a reaction which is reversible in vivo (Buck et al., Biochem.
24:6245-6252 (1985)). Given the structural similarity between succinate
and adipate, that is, both are straight chain dicarboxylic acids, it is
reasonable to expect some activity of the sucCD enzyme on adipyl-CoA. An
enzyme exhibiting adipyl-CoA ligase activity can equivalently carry out
the ATP-generating production of adipate from adipyl-CoA, here using AMP
and PPi as cofactors, when operating in the opposite physiological
direction as depicted in FIG. 1. Exemplary CoA-ligases include the rat
dicarboxylate-CoA ligase for which the sequence is yet uncharacterized
(Vamecq et al., Biochem. J. 230:683-693 (1985)), either of the two
characterized phenylacetate-CoA ligases from P. chrysogenum
(Lamas-Maceiras et al., Biochem. J. 395, 147-155 (2005); Wang et al.,
Biochem. Biophy. Res. Commun. 360:453-458 (2007)), the phenylacetate-CoA
ligase from Pseudomonas putida (Martinez-Blanco et al., J. Biol. Chem.
265:7084-7090 (1990)), and the 6-carboxyhexanoate-CoA ligase from Bacilis
subtilis (Bower et al., J. Bacteriol. 178:4122-4130 (1996)). The protein
sequences for each of these exemplary gene products can be found using
the following GI numbers and/or GenBank identifiers:

[0301] Another option, using phosphotransadipylase/adipate kinase, is
catalyzed by the gene products of buk1, buk2, and ptb from C.
acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et al., J.
Mol. Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptb
gene encodes an enzyme that can convert butyryl-CoA into
butyryl-phosphate, which is then converted to butyrate via either of the
buk gene products with the concomitant generation of ATP. The analogous
set of transformations, that is, conversion of adipyl-CoA to
adipyl-phosphate followed by conversion of adipyl-phosphate to adipate,
can be carried out by the buk1, buk2, and ptb gene products. The protein
sequences for each of these exemplary gene products can be found using
the following GI numbers and/or GenBank identifiers:

[0302] Alternatively, an acetyltransferase capable of transferring the CoA
group from adipyl-CoA to acetate can be applied. Similar transformations
are catalyzed by the gene products of cat1, cat2, and cat3 of Clostridium
kluyveri which have been shown to exhibit succinyl-CoA,
4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity,
respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);
Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). The
protein sequences for each of these exemplary gene products can be found
using the following GI numbers and/or GenBank identifiers:

[0303] Finally, though not as desirable from an energetic standpoint, the
conversion of adipyl-CoA to adipate can also be carried out by an
acyl-CoA hydrolase or equivalently a thioesterase. The top E. coli gene
candidate is tesB (Naggert et al., J. Biol. Chem. 266:11044-11050
(1991)), which shows high similarity to the human acot8, which is a
dicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westin
et al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has also
been characterized in the rat liver (Deana, Biochem. Int. 26:767-773
(1992)). The protein sequences for each of these exemplary gene products
can be found using the following GI numbers and/or GenBank identifiers:

[0305] The above description provides an exemplary adipate synthesis
pathway by way of a reverse adipate degradation pathway.

Example II

Preparation of an Adipate Producing Microbial Organism Having a Reverse
Degradation Pathway

[0306] This example describes the generation of a microbial organism
capable of producing adipate using the reverse degradation pathway.

[0307] Escherichia coli is used as a target organism to engineer a reverse
adipate degradation pathway as shown in FIG. 2. E. coli provides a good
host for generating a non-naturally occurring microorganism capable of
producing adipate. E. coli is amenable to genetic manipulation and is
known to be capable of producing various products, like ethanol, acetic
acid, formic acid, lactic acid, and succinic acid, effectively under
anaerobic or microaerobic conditions.

[0308] To generate an E. coli strain engineered to produce adipate,
nucleic acids encoding the enzymes utilized in the reverse degradation
pathway are expressed in E. coli using well known molecular biology
techniques (see, for example, Sambrook, supra, 2001; Ausubel supra,
1999). In particular, the paaJ (NP--415915.1), paaH
(NP--415913.1), and maoC (NP--415905.1) genes encoding the
succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA
dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities,
respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. In addition, the bcd
(NP--349317.1), etfAB (349315.1 and 349316.1), and sucCD
(NP--415256.1 and AAC73823.1) genes encoding
5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities,
respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. The two sets of plasmids are
transformed into E. coli strain MG1655 to express the proteins and
enzymes required for adipate synthesis via the reverse degradation
pathway.

[0309] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of
reverse degradation pathway genes is corroborated using methods well
known in the art for determining polypeptide expression or enzymatic
activity, including for example, Northern blots, PCR amplification of
mRNA, immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
adipate is confirmed using HPLC, gas chromatography-mass spectrometry
(GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

[0310] Microbial strains engineered to have a functional adipate synthesis
pathway are further augmented by optimization for efficient utilization
of the pathway. Briefly, the engineered strain is assessed to determine
whether any of the exogenous genes are expressed at a rate limiting
level. Expression is increased for any enzymes expressed at low levels
that can limit the flux through the pathway by, for example, introduction
of additional gene copy numbers.

[0311] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of adipate. One modeling
method is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of adipate.
Adaptive evolution also can be used to generate better producers of, for
example, the acetyl-CoA and succinyl-CoA intermediates or the adipate
product. Adaptive evolution is performed to improve both growth and
production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058
(2004); Alper et al., Science 314:1565-1568 (2006)). Based on the
results, subsequent rounds of modeling, genetic engineering and adaptive
evolution can be applied to the adipate producer to further increase
production.

[0312] For large-scale production of adipate, the above reverse
degradation pathway-containing organism is cultured in a fermenter using
a medium known in the art to support growth of the organism under
anaerobic conditions. Fermentations are performed in either a batch,
fed-batch or continuous manner. Anaerobic conditions are maintained by
first sparging the medium with nitrogen and then sealing the culture
vessel, for example, flasks can be sealed with a septum and crimp-cap.
Microaerobic conditions also can be utilized by providing a small hole in
the septum for limited aeration. The pH of the medium is maintained at a
pH of around 7 by addition of an acid, such as H2SO4. The
growth rate is determined by measuring optical density using a
spectrophotometer (600 nm) and the glucose uptake rate by monitoring
carbon source depletion over time. Byproducts such as undesirable
alcohols, organic acids, and residual glucose can be quantified by HPLC
(Shimadzu, Columbia Md.), for example, using an Aminex® series of
HPLC columns (for example, HPX-87 series) (BioRad, Hercules Calif.),
using a refractive index detector for glucose and alcohols, and a UV
detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779
(2005)).

[0313] This example describes the preparation of an adipate producing
microbial organism using a reverse degradation pathway.

[0315] An additional pathway from that described in Examples I and II that
uses acetyl-CoA and succinyl-CoA as precursors for adipate formation and
passes through the metabolic intermediate, 3-oxoadipate, is shown in FIG.
3. The initial two transformations in this pathway are the two terminal
steps of the degradation pathway for aromatic and choloroaromatic
compounds operating in the reverse direction (Kaschabek et al., J.
Bacteriol. 184:207-215 (2002); Nogales et al., Microbiol. 153:357-365
(2007); Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)).
Specifically, the first step forms 3-oxoadipyl CoA by the condensation of
succinyl- and acetyl-CoA. The second step forms 3-oxoadipate and is
reported to be reversible in Pseudomonas sp. Strain B13 (Kaschabek et
al., J. Bacteriol. 184:207-215 (2002)).

[0316] The subsequent steps involve reduction of 3-oxoadipate to
3-hydroxyadipate (conversion of a keto group to hydroxyl group),
dehydration of 3-hydroxyadipate to yield hexa-2-enedioate, and reduction
of hexa-2-enedioate to form adipate. These steps of the pathway are
analogous to the conversion of oxaloacetate into succinate via the
reductive TCA cycle (see FIG. 4). This supports the steps in the pathway
being thermodynamically favorable subject to the presence of appropriate
metabolite concentrations. The final reduction step can be carried out
either biochemically or by employing a chemical catalyst to convert
hexa-2-enedioate into adipate. Chemical hydrogenation can be performed
using Pt catalyst on activated carbon as has been described in (Niu et
al., Biotechnol. Prog. 18:201-211 (2002)).

[0317] The maximum theoretical yield of adipate using this pathway is 0.92
mole per mole glucose consumed, and oxygen is not required for attaining
these yields (see Table 2). The associated energetics are identical to
those of the reverse adipate pathway. Theoretically, ATP formation of up
to 1.55 moles is observed per mole of glucose utilized through this
pathway. The ATP yield improves to approximately 2.47 moles if
phosphoenolpyruvate kinase (PPCK) is assumed to operate in the direction
of ATP generation. Interestingly, the product yield can be increased
further to 1 mole adipate per mole of glucose consumed if chemical
hydrogenation is used for the last step and a 100% efficiency of
catalysis is assumed. In this scenario, up to 1.95 moles of ATP are
formed theoretically without assuming the reverse functionality of PPCK.

[0318] Successfully engineering this pathway involves identifying an
appropriate set of enzymes with sufficient activity and specificity. This
entails identifying an appropriate set of enzymes, cloning their
corresponding genes into a production host, optimizing fermentation
conditions, and assaying for product formation following fermentation. To
engineer a production host for the production of adipate, one or more
exogenous DNA sequence(s) can be expressed in a host microorganism. In
addition, the host microorganism can have endogenous gene(s) functionally
deleted. These modifications allow the production of adipate using
renewable feedstock.

[0319] Described below are a number of biochemically characterized
candidate genes capable of encoding enzymes that catalyze each step of
the 3-oxoadipate pathway for adipate synthesis. Although this method is
described for E. coli, one skilled in the art can apply these teachings
to any other suitable host organism. Specifically, listed below are genes
that are native to E. coli as well as genes in other organisms that can
be applied to catalyze the appropriate transformations when properly
cloned and expressed.

[0321] Referring to FIG. 3, step 2 involves 3-oxoadipyl-CoA transferase.
In this step, 3-oxoadipate is formed by the transfer of the CoA group
from 3-oxoadipyl-CoA to succinate. This activity is reported in a
two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas (Kaschabek et
al., J. Bacteriol. 184:207-215 (2002)). This enzyme catalyzes a
reversible transformation. The protein sequences of exemplary gene
products for subunit A of this complex can be found using the following
GI numbers and/or GenBank identifiers:

[0323] Referring to FIG. 3, step 3 involves 3-oxoadipate reductase. E.
coli has several candidate alcohol dehydrogenases; two that have
analogous functions are malate dehydrogenase (mdh) and lactate
dehydrogenase (ldhA). While it has not been shown that these two enzymes
have broad substrate specificities in E. coli, lactate dehydrogenase from
Ralstonia eutropha has been shown to demonstrate high activities on
substrates of various chain lengths such as lactate, 2-oxobutyrate,
2-oxopentanoate and 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J.
Biochem. 130:329-334 (1983)). An additional non-native enzyme candidate
for this step is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh)
from the human heart which has been cloned and characterized (Marks et
al., J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is particularly
interesting in that it is a dehydrogenase that operates on a
3-hydroxyacid. Given that dehydrogenases are typically reversible, it is
expected that this gene product, or a homlog thereof, will be capable of
reducing a 3-oxoacid, for example, 3-oxoadipate, to the corresponding
3-hydroxyacid, for example, 3-hydroxyadipate. The protein sequences for
each of these exemplary gene products can be found using the following GI
numbers and/or GenBank identifiers:

[0324] Referring to FIG. 3, step 4 involves 3-hydroxyadipate dehydratase.
In this reaction, 3-hydroxyadipate is dehydrated to hexa-2-enedioate.
Although no direct evidence for this enzymatic transformation has been
identified, most dehydratases catalyze the α, β-elimination of
water. This involves activation of the a-hydrogen by an
electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and
removal of the hydroxyl group from the β-position (Martins et al.,
Proc. Natl. Acad. Sci. USA 101:15645-15649 (2004); Buckel and Golding,
FEMS Microbiol. Rev. 22:523-541 (1998)). The protein sequences for
exemplary gene products can be found using the following GI numbers
and/or GenBank identifiers:

[0325] Other good candidates for carrying out this function are the serine
dehydratases. These enzymes catalyze a very similar transformation in the
removal of ammonia from serine as required in this dehydration step. The
protein sequence for exemplary gene product can be found using the
following GI number and/or GenBank identifier:

[0327] Referring to FIG. 3, step 5 involves 2-enoate reductase. The final
step in the 3-oxoadipate pathway is reduction of the double bond in
hexa-3-enedioate to form adipate. Biochemically, this transformation can
be catalyzed by 2-enoate reductase (EC 1.3.1.31) known to catalyze the
NADH-dependent reduction of a wide variety of α, β-unsaturated
carboxylic acids and aldehydes (Rohdich et al., J. Biol. Chem.
276:5779-5787 (2001)). This enzyme is encoded by enr in several species
of Clostridia (Giesel and Simon, Arch. Microbiol. 135:51-57 (1983))
including C. tyrobutyricum and C. thermoaceticum (now called Moorella
thermoaceticum) (Rohdich, et al., J. Biol. Chem. 276:5779-5787 (2001)).
In the recently published genome sequence of C. kluyveri, 9 coding
sequences for enoate reductases have been reported, out of which one has
been characterized (Seedorf et al., Proc. Natl. Acad. Sci. USA
105:2128-2133 (2008)). The enr genes from both C. tyrobutyricum and C.
thermoaceticum have been cloned and sequenced and show 59% identity to
each other. The former gene is also found to have approximately 75%
similarity to the characterized gene in C. kluyveri (Giesel and Simon,
Arch. Microbiol. 135:51-57 (1983)). It has been reported based on these
sequence results that enr is very similar to the dienoyl CoA reductase in
E. coli (fadH) (Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)).
Several gene candidates thus exist for catalyzing this last step in the
3-oxoadipate pathway and have been listed below. The C. thermoaceticum
enr gene has also been expressed in an enzymatically active form in E.
coli (Rohdich et al., supra, 2001). The protein sequences for exemplary
gene products can be found using the following GI numbers and/or GenBank
identifiers:

[0328] The above description provides an exemplary adipate synthesis
pathway by way of an 3-oxoadipate pathway.

Example IV

Preparation of an Adipate Producing Microbial Organism Having a
3-Oxoadipate Pathway

[0329] This example describes the generation of a microbial organism
capable of producing adipate using the 3-oxoadipate pathway.

[0330] Escherichia coli is used as a target organism to engineer the
3-oxoadipate pathway as shown in FIG. 3. E. coli provides a good host for
generating a non-naturally occurring microorganism capable of producing
adipate. E. coli is amenable to genetic manipulation and is known to be
capable of producing various products, like ethanol, acetic acid, formic
acid, lactic acid, and succinic acid, effectively under anaerobic or
microaerobic conditions.

[0331] To generate an E. coli strain engineered to produce adipate,
nucleic acids encoding the enzymes utilized in the 3-oxoadipate pathway
are expressed in E. coli using well known molecular biology techniques
(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). In
particular, the paaJ (NP--415915.1), pcaIJ (AAN69545.1 and
NP--746082.1), and bdh (AAA58352.1) genes encoding the
succinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase,
and 3-oxoadipate reductase activities, respectively, are cloned into the
pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.
In addition, the acnA (P25516.3) and enr (ACA54153.1) genes encoding
3-hydroxyadipate dehydratase and 2-enoate reductase activities,
respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. The two sets of plasmids are
transformed into E. coli strain MG1655 to express the proteins and
enzymes required for adipate synthesis via the 3-oxoadipate pathway.

[0332] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
3-oxoadipate pathway genes for adipate synthesis is corroborated using
methods well known in the art for determining polypeptide expression or
enzymatic activity, including for example, Northern blots, PCR
amplification of mRNA, immunoblotting, and the like. Enzymatic activities
of the expressed enzymes are confirmed using assays specific for the
individual activities. The ability of the engineered E. coli strain to
produce adipate is confirmed using HPLC, gas chromatography-mass
spectrometry (GCMS) and/or liquid chromatography-mass spectrometry
(LCMS).

[0333] Microbial strains engineered to have a functional adipate synthesis
pathway are further augmented by optimization for efficient utilization
of the pathway. Briefly, the engineered strain is assessed to determine
whether any of the exogenous genes are expressed at a rate limiting
level. Expression is increased for any enzymes expressed at low levels
that can limit the flux through the pathway by, for example, introduction
of additional gene copy numbers.

[0334] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of adipate. One modeling
method is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of adipate.
Adaptive evolution also can be used to generate better producers of, for
example, the acetyl-CoA and succinyl-CoA intermediates or the adipate
product. Adaptive evolution is performed to improve both growth and
production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058
(2004); Alper et al., Science 314:1565-1568 (2006)). Based on the
results, subsequent rounds of modeling, genetic engineering and adaptive
evolution can be applied to the adipate producer to further increase
production.

[0335] For large-scale production of adipate, the 3-oxoadipate
pathway-containing organism is cultured in a fermenter using a medium
known in the art to support growth of the organism under anaerobic
conditions. Fermentations are performed in either a batch, fed-batch or
continuous manner. Anaerobic conditions are maintained by first sparging
the medium with nitrogen and then sealing the culture vessel, for
example, flasks can be sealed with a septum and crimp-cap. Microaerobic
conditions also can be utilized by providing a small hole in the septum
for limited aeration. The pH of the medium is maintained at around a pH
of 7 by addition of an acid, such as H2SO4. The growth rate is
determined by measuring optical density using a spectrophotometer (600
nm) and the glucose uptake rate by monitoring carbon source depletion
over time. Byproducts such as undesirable alcohols, organic acids, and
residual glucose can be quantified by HPLC (Shimadzu), for example, using
an Aminex® series of HPLC columns (for example, HPX-87 series)
(BioRad), using a refractive index detector for glucose and alcohols, and
a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779
(2005)).

[0336] This example describes the preparation of an adipate-producing
microbial organism containing a 3-oxidoadipate pathway.

[0338] Adipate synthesis via a combined biological and chemical conversion
process has been previously described. (Niu et al., Biotechnol. Prog.
18:201-211 (2002)) and is shown in FIG. 5. This method is further
described in U.S. Pat. No. 5,487,987. Adipate synthesis through this
route entails introduction of three heterologous genes into E. coli that
can convert dehydroshikimate into cis,cis-muconic acid (Niu et al.,
supra, 2002). A final chemical hydrogenation step leads to the formation
of adipic acid. In this step, the pretreated fermentation broth that
contained 150 mM cis,cis-muconate was mixed with 10% platinum (Pt) on
activated carbon. The hydrogenation reaction was carried out at 3400 KPa
of hydrogen pressure for two and a half hour at 250° C. with
stirring. The calculated adipate yields are shown in Table 3 assuming
either an enzymatic or chemical catalysis step is utilized to convert
cis,cis-muconate into adipate. Under aerobic conditions, an 85% molar
yield of adipate can be obtained if a chemical reaction is employed for
hydrogenation and a 75% molar yield is obtained if an NADH-based
hydrogenase is used.

[0339] Although this is an exemplary method, there are disadvantages of
this method compared to others, such as those described in Examples I-IV.
For example, the first limitation of this method is the lower theoretical
yields compared to the reverse adipate degradation and 3-oxoadipate
pathways. The second limitation is that the ATP yields of this pathway
are negligible. A third limitation of this pathway is that it involves a
dioxygenase, necessitating a supply of oxygen to the bioreactor and
precluding the option of anaerobic fermentation.

[0340] The above description provides an exemplary adipate synthesis
pathway by way of a cis,cis-muconic acid pathway

[0342] Alpha-keto adipate is a known intermediate in lysine biosynthesis
in S. cerevisiae, and this information was used to identify an additional
pathway for adipic acid biosynthesis (see FIG. 6). Conversion of
alpha-ketoglutarate to alpha-ketoadipate is catalyzed by homocitrate
synthase, homoaconitase, and homoisocitrate dehydrogenase as indicated by
dashed arrows in FIG. 6. Conversion of alpha-ketoadipate into
alpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, an
enzyme reported to be found in rat and in human placenta (Suda et al.,
Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.
Biophys. Res. Commun. 77:586-591 (1977). Subsequent steps involve a
dehydratase for the conversion of alpha-hydroxyadipate into
hexa-2-enedioate followed by its reduction to adipic acid. This last step
can be catalyzed either by an enzyme or can take place through a chemical
reaction as described in Example II. Genes encoding the enzymes for the
alpha-ketoadipate pathway are identified as described in Examples I-IV.

[0343] The adipate yields associated with this pathway are shown in Table
4. Because of the loss of two CO2 molecules during the conversion of
acetyl-CoA to adipate, only 67% of the glucose can be converted into
adipate. This is reflected in the molar yields for this pathway under
aerobic conditions. The yields are further reduced in the absence of
oxygen uptake. Also since the maximum ATP yields under anaerobic
conditions are negligible, the engineered organism will have to utilize
additional substrate to form energy for cell growth and maintenance under
such conditions.

[0346] Two additional pathways for adipate synthesis rely on lysine
degradation to form adipate. One pathway starts from alpha-ketoglutarate
to form lysine (pathway non-native to E. coli and found in S.
cerevisiae), and the other uses aspartate as a starting point for lysine
biosynthesis (pathway native to E. coli). FIG. 7 shows adipate formation
from lysine. The maximum theoretical yields for adipate, both in the
presence and absence of oxygen, using the E. coli stoichiometric model
are shown in Tables 5 and 6, with alpha-ketoglutarate and aspartate as
the respective starting points for lysine. The maximum ATP yields
accompanying these theoretical yields were also calculated and are shown
in the same tables. These yields are lower in comparison to the other
pathways described in Examples I-IV. Genes encoding the enzymes for the
alpha-ketoadipate pathway are identified as described in Examples I-IV.

[0349] An exemplary pathway for forming caprolactam and/or 6-aminocaproic
acid using adipyl-CoA as the precursor is shown in FIG. 8. The pathway
involves a CoA-dependant aldehyde dehydrogenase that can reduce
adipyl-CoA to adipate semialdehyde and a transaminase or 6-aminocaproate
dehydrogenase that can transform this molecule into 6-aminocaproic acid.
The terminal step that converts 6-aminocaproate into caprolactam can be
accomplished either via an amidohydrolase or via chemical conversion
(Guit and Buijs, U.S. Pat. No. 6,353,100, issued Mar. 7, 2002; Wolters et
al., U.S. Pat. No. 5,700,934, issued Dec. 23, 1997; Agterberg et al.,
U.S. Pat. No. 6,660,857, issued Dec. 9, 2003). The maximum theoretical
yield of caprolactam was calculated to be 0.8 mole per mole glucose
consumed (see Table 7) assuming that the reverse adipate degradation
pathway was complemented with the reaction scheme shown in FIG. 8. The
pathway is favorable energetically as up to 0.78 moles of ATP are formed
per mole of glucose consumed at the maximum theoretical yield of
caprolactam. The ATP yield can be further improved to 1.63 moles of ATP
produced per mole of glucose if phosphoenolpyruvate carboxykinase (PPCK)
is assumed to function in the ATP-generating direction towards
oxaloacetate formation.

[0350] The final amidohydrolase step is energetically and redox neutral,
and thus the product and ATP molar yields associated with 6-aminocaproic
acid production are equivalent to those associated with caprolactam
production. Thus one can alternatively envision a microorganism and
associated fermentation process that forms 6-aminocaproic acid instead of
caprolactam followed by an additional unit operation to dehydrate/cyclize
6-aminocaproic acid to caprolactam.

[0351] Successfully engineering this pathway involves identifying an
appropriate set of enzymes with sufficient activity and specificity. This
entails identifying an appropriate set of enzymes, cloning their
corresponding genes into a production host, optimizing fermentation
conditions, and assaying for product formation following fermentation. To
engineer a production host for the production of 6-aminocaproic acid or
caprolactam, one or more exogenous DNA sequence(s) can be expressed in a
host microorganism. In addition, the microorganism can have endogenous
gene(s) functionally deleted. These modifications will allow the
production of 6-aminocaproate or caprolactam using renewable feedstock.

[0352] Below is described a number of biochemically characterized
candidate genes capable of encoding enzymes that catalyze each step of
the caprolactam formation pathway described in FIG. 8. Although described
for E. coli, one skilled in the art can apply these teachings to any
other suitable host organism. Specifically, the genes listed are native
to E. coli or are genes in other organisms that can be applied to
catalyze the appropriate transformations when properly cloned and
expressed.

[0354] Referring to FIG. 8, step 2 involves transaminase. The second step
in the pathway is conversion of the 6-aldehyde to an amine This
transformation can likely be accomplished by gamma-aminobutyrate
transaminase (GABA transaminase), a native enzyme encoded by gabT that
transfers an amino group from glutamate to the terminal aldehyde of
succinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042
(1990)). The gene product of puuE catalyzes another 4-aminobutyrate
transaminase in E. coli (Kurihara et al., J. Biol. Chem. 280:4602-4608
(2005)). GABA transaminases in Mus musculus, Pseudomonas fluorescens, and
Sus scrofa have been shown to react with 6-aminocaproic acid (Cooper,
Methods Enzymol. 113:80-82 (1985); Scott and Jakoby, J. Biol. Chem.
234:932-936 (1959)). The protein sequences for exemplary gene products
can be found using the following GI numbers and/or GenBank identifiers:

[0356] Referring to FIG. 8, step 3 involves amidohydrolase. The final step
of caprolactam synthesis is cyclization of 6-aminocaproic acid. This
transformation has not been characterized enzymatically but it is very
similar to the cyclization of lysine by D-lysine lactamase (EC 3.5.2.11)
from Cryptococcus laurentii (Fukumura et al., FEBS Lett. 89:298-300
(1978)). However, the protein and nucleotide sequences of this enzyme are
not currently known and, so far, lysine lactamase activity has not been
demonstrated in other organisms.

[0357] Plasmids contained in several strains of Pseudomonas sp. isolated
from soil have been shown to confer ability to grow on caprolactam as a
sole carbon source (Boronin et al., FEMS Microbiol. Lett. 22:167-170
(1984)); however, associated gene or protein sequences have not been
associated with this function to date.

[0358] The most closely related candidate enzyme with available sequence
information is 6-aminohexanoate-cyclic dimer hydrolase, which has been
characterized in Pseudomonas sp. and Flavobacterium sp. The nylB gene
product from Pseudomonas sp NK87 was cloned and expressed in E. coli
(Kanagawa et al., J. Gen. Microbiol. 139:787-795 (1993)). The substrate
specificity of the enzyme was tested in Flavobacterium sp K172 and was
shown to react with higher-order oligomers of 6-aminohexanoate but not
caprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). The
reversibility and ability of 6-aminohexanoate dimer hydrolases in other
organisms to react with the desired substrate in the direction of
interest can be further tested. The protein sequences for exemplary gene
products can be found using the following GI numbers and/or GenBank
identifiers:

[0359] The above description provides an exemplary pathway to produce
caprolactam and/or 6-aminocaproic acid by way of an adipyl-CoA pathway.

Example IX

Preparation of a 6-Aminocaproate or Caprolactam Producing Microbial
Organism Having a 3-Oxoadipate Pathway

[0360] This example describes the generation of a microbial organism
capable of producing adipate using the reverse degradation pathway and
converting the intracellular adipate to 6-aminocaproate and/or
caprolactam.

[0361] Escherichia coli is used as a target organism to engineer the
necessary genes for adipate, 6-aminocaproate, and/or caprolactam
synthesis (see FIG. 2 and FIG. 8). E. coli provides a good host for
generating a non-naturally occurring microorganism capable of producing
adipate, 6-aminocaproate, and/or caprolactam. E. coli is amenable to
genetic manipulation and is known to be capable of producing various
products, like ethanol, acetic acid, formic acid, lactic acid, and
succinic acid, effectively under anaerobic or microaerobic conditions.

[0362] To generate an E. coli strain engineered to produce 6-aminocaproate
and/or caprolactam, nucleic acids encoding the enzymes utilized in the
reverse adipate degradation pathway and 6-aminocaproate or caprolactam
synthesis pathways are expressed in E. coli using well known molecular
biology techniques (see, for example, Sambrook, supra, 2001; Ausubel,
supra, 1999). In particular, the paaJ (NP--415915.1), paaH
(NP--415913.1), and maoC (NP--415905.1) genes encoding the
succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoA
dehydrogenase, and 3-hydroxyadipyl-CoA dehydratase activities,
respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. In addition, the bcd
(NP--349317.1), etfAB (349315.1 and 349316.1), and sucCD
(NP--415256.1 and AAC73823.1) genes encoding
5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities,
respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. Lastly, the acr1
(YP--047869.1), gabT (NP--417148.1), and nylB (AAA24929.1)
genes encoding CoA-dependent aldehyde dehydrogenase, transaminase, and
amidohydrolase activities are cloned into a third compatible plasmid,
pZS23, under the PA1/lacO promoter. pZS23 is obtained by replacing the
ampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim,
Germany) with a kanamycin resistance module by well-known molecular
biology techniques. The three sets of plasmids are transformed into E.
coli strain MG1655 to express the proteins and enzymes required for
6-aminocaproate and/or caprolactam synthesis.

[0363] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
6-aminocaproate and caprolactam synthesis genes is corroborated using
methods well known in the art for determining polypeptide expression or
enzymatic activity, including for example, Northern blots, PCR
amplification of mRNA, immunoblotting, and the like. Enzymatic activities
of the expressed enzymes are confirmed using assays specific for the
individual activities. The ability of the engineered E. coli strain to
produce 6-aminocaproate and/or caprolactam is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass
spectrometry (LCMS).

[0364] Microbial strains engineered to have a functional pathway for the
synthesis of 6-aminocaproate and/or caprolactam are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the exogenous
genes are expressed at a rate limiting level. Expression is increased for
any enzymes expressed at low levels that can limit the flux through the
pathway by, for example, introduction of additional gene copy numbers.

[0365] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of 6-aminocaproate and/or
caprolactam. One modeling method is the bilevel optimization approach,
OptKnock (Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)),
which is applied to select gene knockouts that collectively result in
better production of 6-aminocaproate and/or caprolactam. Adaptive
evolution also can be used to generate better producers of, for example,
the acetyl-CoA and succinyl-CoA intermediates of the products. Adaptive
evolution is performed to improve both growth and production
characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper
et al., Science 314:1565-1568 (2006)). Based on the results, subsequent
rounds of modeling, genetic engineering and adaptive evolution can be
applied to the 6-aminocaproate and/or caprolactam producer to further
increase production.

[0366] For large-scale production of 6-aminocaproate and/or caprolactam,
the above organism is cultured in a fermenter using a medium known in the
art to support growth of the organism under anaerobic conditions.
Fermentations are performed in either a batch, fed-batch or continuous
manner. Anaerobic conditions are maintained by first sparging the medium
with nitrogen and then sealing the culture vessel, for example, flasks
can be sealed with a septum and crimp-cap. Microaerobic conditions also
can be utilized by providing a small hole in the septum for limited
aeration. The pH of the medium is maintained at around a pH of 7 by
addition of an acid, such as H2SO4. The growth rate is
determined by measuring optical density using a spectrophotometer (600
nm) and the glucose uptake rate by monitoring carbon source depletion
over time. Byproducts such as undesirable alcohols, organic acids, and
residual glucose can be quantified by HPLC (Shimadzu), for example, using
an Aminex® series of HPLC columns (for example, HPX-87 series)
(BioRad), using a refractive index detector for glucose and alcohols, and
a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779
(2005)).

Example X

Adipate Synthesis via 2-Hydroxyadipyl-CoA

[0367] This example describes two exemplary adipate synthesis pathways
proceeding from alpha-ketoadipate and passing through a
2-hydroxyadipyl-CoA intermediate.

[0368] As described in example VI, alpha-ketoadipate is a known
intermediate in lysine biosynthesis that can be formed from
alpha-ketoglutarate via homocitrate synthase, homoaconitase, and
homoisocitrate dehydrogenase. Alpha-ketoadipate can be converted to
2-hydroxyadipyl-CoA by the two routes depicted in FIG. 9.
2-hydroxyadipyl-CoA can be subsequently dehydrated and reduced to
adipyl-CoA which can then be converted to adipate as shown in FIG. 9. The
maximum yield of adipate from glucose via these pathways is 0.67 mol/mol.

[0369] Conversion of alpha-ketoadipate into 2-hydroxyadipate can be
catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in
rat and in human placenta (Suda et al., Arch. Biochem. Biophys.
176:610-620 (1976); Suda et al., Biochem. Biophys. Res. Commun.
77:586-591 (1977). Alternatively, enzymes capable of reducing
alpha-ketoglutarate to 2-hydroxyglutarate may also show activity on
alpha-ketoadipate, which is only one carbon atom longer. One such enzyme
possessing alpha-ketoglutarate reductase activity is serA of Escherichia
coli (Zhao and Winkler, J. Bacteriol. 178(1):232-9 (1996)). Additional
exemplary enzymes can be found in Arabidopsis thaliana (Ho, et al., J.
Biol. Chem. 274(1):397-402 (1999)) and Haemophilus influenzae.

[0370] Referring to FIG. 9, 2-hydroxyadipate can likely be converted to
2-hydroxyadipyl-CoA by the synthetases, transferases,
phosphotransadipylases and kinases described in example I. Alternatively,
enzymes with 2-hydroxyglutarate CoA-transferase or glutaconate
CoA-transferase activity are likely suitable to transfer a CoA moiety to
2-hydroxyadipate. One example of such an enzyme is encoded by the gctA
and gctB genes of Acidaminococcus fermentans (Buckel, et al., Eur. J.
Biochem. 118(2):315-321 (1981); Mack, et al., Eur. J. Biochem.
226(1):41-51 (1994)). Similarly, synthetase, transferase, or
phosphotransadipylase and kinase activities would be required to convert
alpha-ketoadipate into alpha-ketoadipyl-CoA, as depicted in FIG. 9.
Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be carried
out by an alpha-hydroxyacyl-CoA dehydrogenase enzyme. A similar activity
was reported in propionate-adapted E. coli cells whose extracts catalyzed
the oxidation of lactyl-CoA to form pyruvyl-CoA (Megraw et al., J.
Bacteriol. 90(4): 984-988 (1965)). Additional hydroxyacyl-CoA
dehydrogenases were described in example I.

[0372] Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out
by the enzymes described in Example I.

[0373] The above description provides an exemplary adipate synthesis
pathway by way of a 2-hydroxyadipyl-CoA pathway.

Example XI

Preparation of an Adipate Producing Microbial Organism Having a
2-Hydroxyadipyl-CoA Pathway

[0374] This example describes the generation of a microbial organism
capable of producing adipate using a 2-hydroxyadipyl-CoA pathway.

[0375] Escherichia coli is used as a target organism to engineer the
necessary genes for adipate synthesis (see FIG. 9). E. coli provides a
good host for generating a non-naturally occurring microorganism capable
of producing adipate. E. coli is amenable to genetic manipulation and is
known to be capable of producing various products, like ethanol, acetic
acid, formic acid, lactic acid, and succinic acid, effectively under
anaerobic or microaerobic conditions.

[0376] To generate an E. coli strain engineered to produce adipate,
nucleic acids encoding the enzymes utilized in a 2-hydroxyadipyl-CoA to
adipate pathway are expressed in E. coli using well known molecular
biology techniques (see, for example, Sambrook, supra, 2001; Ausubel,
supra, 1999). In particular, the serA (NP--417388.1), gctA (Q59111),
and gctB (Q59112) genes encoding the 2-hydroxyadipate dehydrogenase and
2-hydroxyadipyl-CoA:acetyl-CoA transferase activities, respectively, are
cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the
PA1/lacO promoter. In addition, the hgdA (P11569), hgdB (P11570), and
hgdC (P11568) genes encoding 2-hydroxyadipyl-CoA dehydratase activity,
respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. Further, the bcd
(NP--349317.1), etfAB (349315.1 and 349316.1), and sucCD
(NP--415256.1 and AAC73823.1) genes encoding
5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase activities
are cloned into a third compatible plasmid, pZS23, under the PA1/lacO
promoter. pZS23 is obtained by replacing the ampicillin resistance module
of the pZS13 vector (Expressys, Ruelzheim, Germany) with a kanamycin
resistance module by well-known molecular biology techniques. The three
sets of plasmids are transformed into E. coli strain MG1655 to express
the proteins and enzymes required for adipate synthesis.

[0377] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
2-hydroxyadipyl-CoA pathway genes for adipate synthesis is corroborated
using methods well known in the art for determining polypeptide
expression or enzymatic activity, including for example, Northern blots,
PCR amplification of mRNA, immunoblotting, and the like. Enzymatic
activities of the expressed enzymes are confirmed using assays specific
for the individual activities. The ability of the engineered E. coli
strain to produce adipate is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) and/or liquid chromatography-mass
spectrometry (LCMS).

[0378] Microbial strains engineered to have a functional adipate synthesis
pathway are further augmented by optimization for efficient utilization
of the pathway. Briefly, the engineered strain is assessed to determine
whether any of the exogenous genes are expressed at a rate limiting
level. Expression is increased for any enzymes expressed at low levels
that can limit the flux through the pathway by, for example, introduction
of additional gene copy numbers.

[0379] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of adipate. One modeling
method is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of adipate.
Adaptive evolution also can be used to generate better producers of, for
example, the alpha-ketoadipate intermediate or the adipate product.
Adaptive evolution is performed to improve both growth and production
characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper
et al., Science 314:1565-1568 (2006)). Based on the results, subsequent
rounds of modeling, genetic engineering and adaptive evolution can be
applied to the adipate producer to further increase production.

[0380] For large-scale production of adipate, the 2-hydroxyadipyl-CoA
pathway-containing organism is cultured in a fermenter using a medium
known in the art to support growth of the organism under anaerobic
conditions. Fermentations are performed in either a batch, fed-batch or
continuous manner. Anaerobic conditions are maintained by first sparging
the medium with nitrogen and then sealing the culture vessel, for
example, flasks can be sealed with a septum and crimp-cap. Microaerobic
conditions also can be utilized by providing a small hole in the septum
for limited aeration. The pH of the medium is maintained at around a pH
of 7 by addition of an acid, such as H2SO4. The growth rate is
determined by measuring optical density using a spectrophotometer (600
nm) and the glucose uptake rate by monitoring carbon source depletion
over time. Byproducts such as undesirable alcohols, organic acids, and
residual glucose can be quantified by HPLC (Shimadzu), for example, using
an Aminex® series of HPLC columns (for example, HPX-87 series)
(BioRad), using a refractive index detector for glucose and alcohols, and
a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779
(2005)).

[0381] This example describes the preparation of an adipate-producing
microbial organism containing a 2-hydroxyadipyl-CoA pathway.

Example XII

Pathways for Production of Hexamethylenediamine, Caprolactam and
6-Aminocaproic Acid

[0382] This example describes exemplary pathways for production of
hexamethylenediamine, caprolactam and 6-aminocaproic acid.

[0383] Described below are various pathways leading to the production of
caprolactam, hexamethylenediamine (HMDA), or 6-aminocaproate from common
central metabolites. The first described pathway entails the activation
of 6-aminocaproate to 6-aminocaproyl-CoA by a transferase or synthase
enzyme (FIG. 10, Step Q or R) followed by the spontaneous cyclization of
6-aminocaproyl-CoA to form caprolactam (FIG. 10, Step T). The second
described pathway entails the activation of 6-aminocaproate to
6-aminocaproyl-CoA (FIG. 10, Step Q or R), followed by a reduction (FIG.
10, Step U) and amination (FIG. 10, Step V or W) to form HMDA.
6-Aminocaproic acid can alternatively be activated to
6-aminocaproyl-phosphate instead of 6-aminocaproyl-CoA.
6-Aminocaproyl-phosphate can spontaneously cyclize to form caprolactam.
Alternatively, 6-aminocaproyl-phosphate can be reduced to 6-aminocaproate
semialdehye, which can be then converted to HMDA as depicted in FIGS. 10
and 11. In either this case, the amination reaction must occur relatively
quickly to minimize the spontaneous formation of the cyclic imine of
6-aminocaproate semialdehyde. Linking or scaffolding the participating
enzymes represents a potentially powerful option for ensuring that the
6-aminocaproate semialdehyde intermediate is efficiently channeled from
the reductase enzyme to the amination enzyme.

[0384] Another option for minimizing or even eliminating the formation of
the cyclic imine or caprolactam during the conversion of 6-aminocaproic
acid to HMDA entails adding a functional group (for example, acetyl,
succinyl) to the amine group of 6-aminocaproic acid to protect it from
cyclization. This is analogous to ornithine formation from L-glutamate in
Escherichia coli. Specifically, glutamate is first converted to
N-acetyl-L-glutamate by N-acetylglutamate synthase. N-Acetyl-L-glutamate
is then activated to N-acetylglutamyl-phosphate, which is reduced and
transaminated to form N-acetyl-L-ornithine. The acetyl group is then
removed from N-acetyl-L-ornithine by N-acetyl-L-ornithine deacetylase
forming L-ornithine. Such a route is necessary because formation of
glutamate-5-phosphate from glutamate followed by reduction to
glutamate-5-semialdehyde leads to the formation of
(S)-1-pyrroline-5-carboxylate, a cyclic imine formed spontaneously from
glutamate-5-semialdehyde. In the case of forming HMDA from 6-aminocaproic
acid, the steps can involve acetylating 6-aminocaproic acid to
acetyl-6-aminocaproic acid, activating the carboxylic acid group with a
CoA or phosphate group, reducing, aminating, and deacetylating.

[0385] Note that 6-aminocaproate can be formed from various starting
molecules. For example, the carbon backbone of 6-aminocaproate can be
derived from succinyl-CoA and acetyl-CoA as depicted in FIG. 10 and also
described in FIGS. 2, 3 and 8. Alternatively, 6-aminocaproate can be
derived from alpha-ketoadipate, where alpha-ketoadipate is converted to
adipyl-CoA (see FIG. 9), and adipyl-CoA is converted to 6-aminocaproate
as shown in FIG. 10.

[0386] FIG. 11 provides two additional metabolic pathways to
6-aminocaproate or 6-aminocapropyl-CoA starting from 4-aminobutyryl-CoA
and acetyl-CoA. The first route entails the condensation of
4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (Step
A) followed by a reduction (Step B), dehydration (Step C), and reduction
(Step D) to form 6-aminocaproyl-CoA. 6-Aminocaproyl-CoA can be converted
to 6-aminocaproate by a transferase (Step K), synthase (Step L), or
hydrolase (Step M) enzyme. Alternatively, 6-aminocaproyl-CoA can be
converted to caprolactam by spontaneous cyclization (Step Q) or to HMDA
following its reduction (Step N) and amination (Step O or P). The second
pathway described in FIG. 11 entails the condensation of
4-aminobutyryl-CoA and acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (Step
A) which is then converted to 3-oxo-6-aminohexanoate by a transferase
(Step E), synthase (Step F), or hydrolase (Step G).
3-Oxo-6-aminohexanoate is then reduced (Step H), dehydrated (Step I), and
reduced (Step J) to form 6-aminocaproate.

[0387] The starting molecule, 4-aminobutyryl-CoA, can be formed from
various common central metabolites. For example, glutamate can be
decarboxylated to 4-aminobutyrate, which is then activated by a
CoA-transferase or synthase to 4-aminobutyryl-CoA. Alternatively,
succinate semialdehyde, formed from either the reduction of succinyl-CoA
or the decarboxylation of alpha-ketoglutarate, can be transaminated to
4-aminobutyrate prior to activation by a CoA-transferase or synthase to
form 4-aminobutyryl-CoA. It is noted that 4-aminobutyryl-CoA and several
of the intermediates of the 4-aminobutyryl-CoA to 6-aminocaproyl-CoA
pathway may spontaneously cyclize to their corresponding lactams. Thus,
adding a protective functional group to the terminal amine group of
4-aminobutyryl-CoA and/or several of the amino-CoA intermediates can be
used to minimize the formation of unwanted cyclic byproducts. In this
case, the same general set of transformations depicted in FIG. 11 would
apply, although two additional steps, for example, an acetylase and
deacetylase, can be added to the pathway.

[0388] All transformations depicted in FIGS. 10-11 fall into the 12
general categories of transformations shown in Table 8. Below is
described a number of biochemically characterized candidate genes in each
category. Specifically listed are genes that can be applied to catalyze
the appropriate transformations in FIGS. 10-11 when cloned and expressed.

[0390] Four transformations depicted in FIGS. 10 and 11 require
oxidoreductases that convert a ketone functionality to a hydroxyl group.
Step B in both FIGS. 10 and 11 involves converting a 3-oxoacyl-CoA to a
3-hydroxyacyl-CoA. Step H in both FIGS. 1 and 2 involves converting a
3-oxoacid to a 3-hydroxyacid.

[0391] Exemplary enzymes that can convert 3-oxoacyl-CoA molecules such as
3-oxoadipyl-CoA and 3-oxo-6-aminohexanoyl-CoA into 3-hydroxyacyl-CoA
molecules such as 3-hydroxyadipyl-CoA and 3-hydroxy-6-aminohexanoyl-CoA,
respectively, include enzymes whose natural physiological roles are in
fatty acid beta-oxidation or phenylacetate catabolism. For example,
subunits of two fatty acid oxidation complexes in E. coli, encoded by
fadB and fadJ, function as 3-hydroxyacyl-CoA dehydrogenases (Binstock et
al., Methods Enzymol. 71:403-411 (1981)). Furthermore, the gene products
encoded by phaC in Pseudomonas putida U (Olivera et al., Proc. Natl.
Acad. Sci. USA 95:6419-6424 (1998)) and paaC in Pseudomonas fluorescens
ST (Di Gennaro et al., Arch. Microbiol. 188:117-125 (2007)) catalyze the
reverse reaction of step B in FIG. 10, that is, the oxidation of
3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during the catabolism of
phenylacetate or styrene. Note that the reactions catalyzed by such
enzymes are reversible. In addition, given the proximity in E. coli of
paaH to other genes in the phenylacetate degradation operon (Nogales et
al., Microbiology 153:357-365 (2007)) and the fact that paaH mutants
cannot grow on phenylacetate (Ismail et al., Eur. J. Biochem.
270:3047-3054 (2003)), it is expected that the E. coli paaH gene encodes
a 3-hydroxyacyl-CoA dehydrogenase.

[0397] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle
in thermoacidophilic archaeal bacteria (Berg et al., supra; Thauer R. K.,
Science 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor
and has been characterized in Metallosphaera and Sulfolobus spp (Alber et
al., J. Bacteriol. 188:8551-8559 (2006); Hugler et al., J. Bacteriol.
184:2404-2410 (2002)). The enzyme is encoded by Msed--0709 in
Metallosphaera sedula (Alber et al., supra; Berg et al., supra). A gene
encoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned and
heterologously expressed in E. coli (Alber et al., supra). This enzyme
has also been shown to catalyze the conversion of methylmalonyl-CoA to
its corresponding aldehyde (WO/2007/141208). Although the aldehyde
dehydrogenase functionality of these enzymes is similar to the
bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little
sequence similarity. Both malonyl-CoA reductase enzyme candidates have
high sequence similarity to aspartate-semialdehyde dehydrogenase, an
enzyme catalyzing the reduction and concurrent dephosphorylation of
aspartyl-4-phosphate to aspartate semialdehyde. Additional gene
candidates can be found by sequence homology to proteins in other
organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius
and have been listed below. Yet another candidate for CoA-acylating
aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii
(Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme
has been reported to reduce acetyl-CoA and butyryl-CoA to their
corresponding aldehydes. This gene is very similar to cutE that encodes
acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et
al., supra).

[0399] Referring to FIG. 10, step D refers to the conversion of
5-carboxy-2-pentenoyl-CoA to adipyl-CoA by 5-carboxy-2-pentenoyl-CoA
reductase. Referring to FIG. 11, step D refers to the conversion of
6-aminohex-2-enoyl-CoA to 6-aminocaproyl-CoA. Enoyl-CoA reductase enzymes
are suitable enzymes for either transformation. One exemplary enoyl-CoA
reductase is the gene product of bcd from C. acetobutylicum (Boynton et
al., J Bacteriol. 178:3015-3024 (1996); Atsumi et al., Metab. Eng. 2008
10(6):305-311 (2008) (Epub Sep. 14, 2007), which naturally catalyzes the
reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can be
enhanced by expressing bcd in conjunction with expression of the C.
acetobutylicum etfAB genes, which encode an electron transfer
flavoprotein. An additional candidate for the enoyl-CoA reductase step is
the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et
al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this
sequence following the removal of its mitochondrial targeting leader
sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister
et al., supra). This approach is well known to those skilled in the art
of expressing eukaryotic genes, particularly those with leader sequences
that may target the gene product to a specific intracellular compartment,
in prokaryotic organisms. A close homolog of this gene, TDE0597, from the
prokaryote Treponema denticola represents a third enoyl-CoA reductase
which has been cloned and expressed in E. coli (Tucci et al., FEBS
Letters 581:1561-1566 (2007)).

[0400] Step J of both FIGS. 10 and 11 requires a 2-enoate reductase
enzyme. 2-Enoate reductases (EC 1.3.1.31) are known to catalyze the
NAD(P)H-dependent reduction of a wide variety of α,
β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J.
Biol. Chem. 276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr
in several species of Clostridia (Giesel et al., Arch Microbiol 135:51-57
(1983)) including C. tyrobutyricum, and C. thermoaceticum (now called
Moorella thermoaceticum) (Rohdich et al., supra). In the published genome
sequence of C. kluyveri, 9 coding sequences for enoate reductases have
been reported, out of which one has been characterized (Seedorf et al.,
Proc. Natl. Acad. Sci. USA, 105:2128-2133 (2008)). The enr genes from
both C. tyrobutyricum and C. thermoaceticum have been cloned and
sequenced and show 59% identity to each other. The former gene is also
found to have approximately 75% similarity to the characterized gene in
C. kluyveri (Giesel et al., supra). It has been reported based on these
sequence results that enr is very similar to the dienoyl CoA reductase in
E. coli (fadH) (Rohdich et al., supra). The C. thermoaceticum enr gene
has also been expressed in an enzymatically active form in E. coli
(Rohdich et al., supra).

[0406] Referring to FIG. 10, step A involves 3-oxoadipyl-CoA thiolase, or
equivalently, succinyl CoA:acetyl CoA acyl transferase
(β-ketothiolase). The gene products encoded by pcaF in Pseudomonas
strain B13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)), phaD in
Pseudomonas putida U (Olivera et al., supra), paaE in Pseudomonas
fluorescens ST (Di Gennaro et al., supra), and paaJ from E. coli (Nogales
et al., supra) catalyze the conversion of 3-oxoadipyl-CoA into
succinyl-CoA and acetyl-CoA during the degradation of aromatic compounds
such as phenylacetate or styrene. Since β-ketothiolase enzymes
catalyze reversible transformations, these enzymes can be employed for
the synthesis of 3-oxoadipyl-CoA. For example, the ketothiolase phaA from
R. eutropha combines two molecules of acetyl-CoA to form acetoacetyl-CoA
(Sato et al., J Biosci Bioeng 103:38-44 (2007)). Similarly, a β-keto
thiolase (bktB) has been reported to catalyze the condensation of
acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA (Slater et
al., J. Bacteriol. 180:1979-1987 (1998)) in R. eutropha. In addition to
the likelihood of possessing 3-oxoadipyl-CoA thiolase activity, all such
enzymes represent good candidates for condensing 4-aminobutyryl-CoA and
acetyl-CoA to form 3-oxo-6-aminohexanoyl-CoA (step A, FIG. 11) either in
their native forms or once they have been appropriately engineered.

[0410] Additional enzyme candidates include putrescine aminotransferases
or other diamine aminotransferases. Such enzymes are particularly well
suited for carrying out the conversion of 6-aminocaproate semialdehyde to
hexamethylenediamine The E. coli putrescine aminotransferase is encoded
by the ygjG gene and the purified enzyme also was able to transaminate
cadaverine and spermidine (Samsonova et al., BMC Microbiol 3:2 (2003)).
In addition, activity of this enzyme on 1,7-diaminoheptane and with amino
acceptors other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has
been reported (Samsonova et al., supra; Kim, K. H., J Biol Chem
239:783-786 (1964)). A putrescine aminotransferase with higher activity
with pyruvate as the amino acceptor than alpha-ketoglutarate is the spuC
gene of Pseudomonas aeruginosa (Lu et al., J Bacteriol 184:3765-3773
(2002)).

[0413] CoA transferases catalyze reversible reactions that involve the
transfer of a CoA moiety from one molecule to another. For example, step
E of FIG. 10 is catalyzed by a 3-oxoadipyl-CoA transferase. In this step,
3-oxoadipate is formed by the transfer of the CoA group from
3-oxoadipyl-CoA to succinate, acetate, or another CoA acceptor. Step E of
FIG. 11 entails the transfer of a CoA moiety from another 3-oxoacyl-CoA,
3-oxo-6-aminohexanoyl-CoA. One candidate enzyme for these steps is the
two-unit enzyme encoded by pcaI and pcaJ in Pseudomonas, which has been
shown to have 3-oxoadipyl-CoA/succinate transferase activity (Kaschabek
et al., supra). Similar enzymes based on homology exist in Acinetobacter
sp. ADP1 (Kowalchuk et al., Gene 146:23-30 (1994)) and Streptomyces
coelicolor. Additional exemplary succinyl-CoA:3:oxoacid-CoA transferases
are present in Helicobacter pylori (Corthesy-Theulaz et al., J. Biol.
Chem. 272:25659-25667 (1997)) and Bacillus subtilis (Stols et al.,
Protein. Expr. Purif. 53:396-403 (2007)).

[0425] Most dehydratases catalyze the α, β-elimination of
water. This involves activation of the a-hydrogen by an
electron-withdrawing carbonyl, carboxylate, or CoA-thiol ester group and
removal of the hydroxyl group from the β-position. Enzymes
exhibiting activity on substrates with an electron-withdrawing
carboxylate group are excellent candidates for dehydrating
3-hydroxyadipate (FIG. 10, Step I) or 3-hydroxy-6-aminohexanoate (FIG.
11, Step I).

[0429] An additional enzyme candidate is 2-methylmalate dehydratase, also
called citramalate hydrolyase, a reversible hydrolyase that catalyzes the
alpha, beta elimination of water from citramalate to form mesaconate.
This enzyme has been purified and characterized in Clostridium
tetanomorphum (Wang et al., J Biol. Chem. 244:2516-2526 (1969)). The
activity of this enzyme has also been detected in several bacteria in the
genera Citrobacter and Morganella in the context of the glutamate
degradation VI pathway (Kato et al., Arch. Microbiol 168:457-463 (1997)).
Genes encoding this enzyme have not been identified in any organism to
date.

[0432] Steps F, L, and R of FIG. 10 and Steps F and L of FIG. 11 require
acid-thiol ligase or synthetase functionality (the terms ligase,
synthetase, and synthase are used herein interchangeably and refer to the
same enzyme class). Exemplary genes encoding enzymes likely to carry out
these transformations include the sucCD genes of E. coli which naturally
form a succinyl-CoA synthetase complex. This enzyme complex naturally
catalyzes the formation of succinyl-CoA from succinate with the
concaminant consumption of one ATP, a reaction which is reversible in
vivo (Buck et al., Biochem. 24:6245-6252 (1985)). Given the structural
similarity between succinate and adipate, that is, both are straight
chain dicarboxylic acids, it is reasonable to expect some activity of the
sucCD enzyme on adipyl-CoA.

[0435] Yet another option is to employ a set of enzymes with net ligase or
synthetase activity. For example, phosphotransadipylase and adipate
kinase enzymes are catalyzed by the gene products of buk1, buk2, and ptb
from C. acetobutylicum (Walter et al., Gene 134:107-111 (1993); Huang et
al., J. Mol. Microbiol. Biotechnol. 2:33-38 (2000)). The ptb gene encodes
an enzyme that can convert butyryl-CoA into butyryl-phosphate, which is
then converted to butyrate via either of the buk gene products with the
concomitant generation of ATP.

[0436] No enzyme required--Spontaneous cyclization. 6-Aminocaproyl-CoA
will cyclize spontaneously to caprolactam, thus eliminating the need for
a dedicated enzyme for this step. A similar spontaneous cyclization is
observed with 4-aminobutyryl-CoA which forms pyrrolidinone (Ohsugi et
al., J Biol Chem 256:7642-7651 (1981)).

Example XIII

Preparation of a 6-Aminocaproic Acid Producing Microbial Organism Having a
Pathway for Converting Acetyl-CoA and 4-Aminobutyryl-CoA to
6-Aminocaproic Acid

[0437] This example describes the generation of a microbial organism
capable of producing 6-aminocaproic acid from acetyl-CoA and
4-aminobutyryl-CoA.

[0438] Escherichia coli is used as a target organism to engineer the
6-aminocaproic acid pathway shown in FIG. 11 that starts from acetyl-CoA
and 4-aminobutyryl-CoA. E. coli provides a good host for generating a
non-naturally occurring microorganism capable of producing 6-aminocaproic
acid. E. coli is amenable to genetic manipulation and is known to be
capable of producing various products, like ethanol, acetic acid, formic
acid, lactic acid, and succinic acid, effectively under anaerobic or
microaerobic conditions.

[0439] To generate an E. coli strain engineered to produce 6-aminocaproic
acid, nucleic acids encoding the requisite enzymes are expressed in E.
coli using well known molecular biology techniques (see, for example,
Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ
(NP--415915.1), paaH (NP--415913.1), and maoC
(NP--415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA
thiolase, 3-oxo-6-aminohexanoyl-CoA reductase,
3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are
cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the
PA1/lacO promoter. In addition, the bcd (NP--349317.1), etfAB
(NP--349315.1 and NP--349316.1), and acot8 (CAA15502) genes
encoding 6-aminohex-2-enoyl-CoA reductase and 6-aminocaproyl-CoA
hydrolase activities are cloned into the pZA33 vector (Expressys,
Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the sucD
(NP--904963.1), gabT (NP--417148.1), and cat2 (P38942.2) genes
encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase,
and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a
third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase
the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing
the ampicillin resistance module of the pZS13 vector (Expressys,
Ruelzheim, Germany) with a kanamycin resistance module by well-known
molecular biology techniques. The three sets of plasmids are transformed
into E. coli strain MG1655 to express the proteins and enzymes required
for 6-aminocaproic acid synthesis.

[0440] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
6-aminocaproic acid synthesis genes is corroborated using methods well
known in the art for determining polypeptide expression or enzymatic
activity, including for example, Northern blots, PCR amplification of
mRNA, immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass
spectrometry (GCMS) and/or liquid chromatography-mass spectrometry
(LCMS).

[0441] Microbial strains engineered to have a functional 6-aminocaproic
acid synthesis pathway are further augmented by optimization for
efficient utilization of the pathway. Briefly, the engineered strain is
assessed to determine whether any of the exogenous genes are expressed at
a rate limiting level. Expression is increased for any enzymes expressed
at low levels that can limit the flux through the pathway by, for
example, introduction of additional gene copy numbers.

[0442] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of 6-aminocaproic acid. One
modeling method is the bilevel optimization approach, OptKnock (Burgard
et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to
select gene knockouts that collectively result in better production of
6-aminocaproic acid. Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA and succinyl-CoA
intermediates of the 6-aminocaproic acid product. Adaptive evolution is
performed to improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied to
the 6-aminocaproic acid producer to further increase production.

[0443] For large-scale production of 6-aminocaproic acid, the above
organism is cultured in a fermenter using a medium known in the art to
support growth of the organism under anaerobic conditions. Fermentations
are performed in either a batch, fed-batch or continuous manner.
Anaerobic conditions are maintained by first sparging the medium with
nitrogen and then sealing the culture vessel, for example, flasks can be
sealed with a septum and crimp-cap. Microaerobic conditions also can be
utilized by providing a small hole in the septum for limited aeration.
The pH of the medium is maintained at a pH of around 7 by addition of an
acid, such as H2SO4. The growth rate is determined by measuring
optical density using a spectrophotometer (600 nm) and the glucose uptake
rate by monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XIV

Preparation of a 6-Aminocaproic Acid Producing Microbial Organism Having a
Pathway for Converting Acetyl-CoA and 4-Aminobutyryl-CoA to
6-Aminocaproic Acid

[0444] This example describes the generation of a microbial organism
capable of producing 6-aminocaproic acid from acetyl-CoA and
4-aminobutyryl-CoA.

[0445] Escherichia coli is used as a target organism to engineer the
6-aminocaproic acid pathway shown in FIG. 11 that starts from acetyl-CoA
and 4-aminobutyryl-CoA. E. coli provides a good host for generating a
non-naturally occurring microorganism capable of producing 6-aminocaproic
acid. E. coli is amenable to genetic manipulation and is known to be
capable of producing various products, like ethanol, acetic acid, formic
acid, lactic acid, and succinic acid, effectively under anaerobic or
microaerobic conditions.

[0446] To generate an E. coli strain engineered to produce 6-aminocaproic
acid, nucleic acids encoding the requisite enzymes are expressed in E.
coli using well known molecular biology techniques (see, for example,
Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ
(NP--415915.1), pcaIJ (AAN69545.1 and NP--746082.1), and bdh
(AAA58352.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA thiolase,
3-oxo-6-aminohexanoyl-CoA/acyl-CoA transferase, 3-oxo-6-aminohexanoate
reductase activities, respectively, are cloned into the pZE13 vector
(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. In addition,
the enr (CAA76083.1) and hmd (ABC88407.1) genes encoding
6-aminohex-2-enoate reductase and 3-hydroxy-6-aminohexanoate dehydratase
activities are cloned into the pZA33 vector (Expressys, Ruelzheim,
Germany) under the PA1/lacO promoter. Lastly, the sucD
(NP--904963.1), gabT (NP--417148.1), and cat2 (P38942.2) genes
encoding succinyl-CoA reductase (aldehyde forming), GABA transaminase,
and 4-aminobutyryl-CoA/acyl-CoA transferase activities are cloned into a
third compatible plasmid, pZS23, under the PA1/lacO promoter, to increase
the availability of 4-aminobutyryl-CoA. pZS23 is obtained by replacing
the ampicillin resistance module of the pZS13 vector (Expressys,
Ruelzheim, Germany) with a kanamycin resistance module by well-known
molecular biology techniques. The three sets of plasmids are transformed
into E. coli strain MG1655 to express the proteins and enzymes required
for 6-aminocaproic acid synthesis.

[0447] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
6-aminocaproic acid synthesis genes is corroborated using methods well
known in the art for determining polypeptide expression or enzymatic
activity, including for example, Northern blots, PCR amplification of
mRNA, immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
6-aminocaproic acid is confirmed using HPLC, gas chromatography-mass
spectrometry (GCMS) and/or liquid chromatography-mass spectrometry
(LCMS).

[0448] Microbial strains engineered to have a functional 6-aminocaproic
acid synthesis pathway are further augmented by optimization for
efficient utilization of the pathway. Briefly, the engineered strain is
assessed to determine whether any of the exogenous genes are expressed at
a rate limiting level. Expression is increased for any enzymes expressed
at low levels that can limit the flux through the pathway by, for
example, introduction of additional gene copy numbers.

[0449] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of 6-aminocaproic acid. One
modeling method is the bilevel optimization approach, OptKnock (Burgard
et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to
select gene knockouts that collectively result in better production of
6-aminocaproic acid. Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA and succinyl-CoA
intermediates of the 6-aminocaproic acid product. Adaptive evolution is
performed to improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied to
the 6-aminocaproic acid producer to further increase production.

[0450] For large-scale production of 6-aminocaproic acid, the above
organism is cultured in a fermenter using a medium known in the art to
support growth of the organism under anaerobic conditions. Fermentations
are performed in either a batch, fed-batch or continuous manner.
Anaerobic conditions are maintained by first sparging the medium with
nitrogen and then sealing the culture vessel, for example, flasks can be
sealed with a septum and crimp-cap. Microaerobic conditions also can be
utilized by providing a small hole in the septum for limited aeration.
The pH of the medium is maintained at a pH of around 7 by addition of an
acid, such as H2SO4. The growth rate is determined by measuring
optical density using a spectrophotometer (600 nm) and the glucose uptake
rate by monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XV

Preparation of a Caprolactam Producing Microbial Organism Having a Pathway
for Converting Acetyl-CoA and Succinyl-CoA to 6-Aminocaproic Acid

[0451] This example describes the generation of a microbial organism
capable of producing caprolactam from acetyl-CoA and succinyl-CoA.

[0452] Escherichia coli is used as a target organism to engineer the
caprolactam pathway shown in FIG. 10 that starts from acetyl-CoA and
succinyl-CoA. E. coli provides a good host for generating a non-naturally
occurring microorganism capable of producing caprolactam. E. coli is
amenable to genetic manipulation and is known to be capable of producing
various products, like ethanol, acetic acid, formic acid, lactic acid,
and succinic acid, effectively under anaerobic or microaerobic
conditions.

[0453] To generate an E. coli strain engineered to produce caprolactam,
nucleic acids encoding the requisite enzymes are expressed in E. coli
using well known molecular biology techniques (see, for example,
Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ
(NP--415915.1), paaH (NP--415913.1), and maoC
(NP--415905.1) genes encoding the 3-oxoadipyl-CoA thiolase,
3-oxoadipyl-CoA reductase, and 3-hydroxyadipyl-CoA dehydratase
activities, respectively, are cloned into the pZE13 vector (Expressys,
Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the bcd
(NP--349317.1) and etfAB (NP--349315.1 and NP--349316.1)
genes encoding 5-carboxy-2-pentenoyl-CoA reductase activity are cloned
into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO
promoter. Lastly, the acr1 (YP--047869.1), gabT (NP--417148.1),
and bioW (NP--390902.2) genes encoding adipyl-CoA reductase
(aldehyde forming), 6-aminocaproic acid transaminase, and
6-aminocaproyl-CoA synthase activities are cloned into a third compatible
plasmid, pZS23, under the PA1/lacO promoter. pZS23 is obtained by
replacing the ampicillin resistance module of the pZS13 vector
(Expressys, Ruelzheim, Germany) with a kanamycin resistance module by
well-known molecular biology techniques. The three sets of plasmids are
transformed into E. coli strain MG1655 to express the proteins and
enzymes required for caprolactam synthesis.

[0454] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
caprolactam synthesis genes is corroborated using methods well known in
the art for determining polypeptide expression or enzymatic activity,
including for example, Northern blots, PCR amplification of mRNA,
immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry
(GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

[0455] Microbial strains engineered to have a functional caprolactam
synthesis pathway are further augmented by optimization for efficient
utilization of the pathway. Briefly, the engineered strain is assessed to
determine whether any of the exogenous genes are expressed at a rate
limiting level. Expression is increased for any enzymes expressed at low
levels that can limit the flux through the pathway by, for example,
introduction of additional gene copy numbers.

[0456] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of caprolactam. One modeling
method is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of
caprolactam. Adaptive evolution also can be used to generate better
producers of, for example, the acetyl-CoA and succinyl-CoA intermediates
of the caprolactam product. Adaptive evolution is performed to improve
both growth and production characteristics (Fong and Palsson, Nat. Genet.
36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based
on the results, subsequent rounds of modeling, genetic engineering and
adaptive evolution can be applied to the caprolactam producer to further
increase production.

[0457] For large-scale production of caprolactam, the above organism is
cultured in a fermenter using a medium known in the art to support growth
of the organism under anaerobic conditions. Fermentations are performed
in either a batch, fed-batch or continuous manner. Anaerobic conditions
are maintained by first sparging the medium with nitrogen and then
sealing the culture vessel, for example, flasks can be sealed with a
septum and crimp-cap. Microaerobic conditions also can be utilized by
providing a small hole in the septum for limited aeration. The pH of the
medium is maintained at a pH of around 7 by addition of an acid, such as
H2SO4. The growth rate is determined by measuring optical
density using a spectrophotometer (600 nm) and the glucose uptake rate by
monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XVI

Preparation of a Hexamethylenediamine Producing Microbial Organism Having
a Pathway for Converting Acetyl-CoA and Succinyl-CoA to 6-Aminocaproic
Acid

[0458] This example describes the generation of a microbial organism
capable of producing hexamethylenediamine from acetyl-CoA and
succinyl-CoA.

[0459] Escherichia coli is used as a target organism to engineer the
hexamethylenediamine pathway shown in FIG. 10 that starts from acetyl-CoA
and succinyl-CoA. E. coli provides a good host for generating a
non-naturally occurring microorganism capable of producing
hexamethylenediamine E. coli is amenable to genetic manipulation and is
known to be capable of producing various products, like ethanol, acetic
acid, formic acid, lactic acid, and succinic acid, effectively under
anaerobic or microaerobic conditions.

[0461] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
hexamethylenediamine synthesis genes is corroborated using methods well
known in the art for determining polypeptide expression or enzymatic
activity, including for example, Northern blots, PCR amplification of
mRNA, immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
hexamethylenediamine is confirmed using HPLC, gas chromatography-mass
spectrometry (GCMS) and/or liquid chromatography-mass spectrometry
(LCMS).

[0462] Microbial strains engineered to have a functional
hexamethylenediamine synthesis pathway are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the exogenous
genes are expressed at a rate limiting level. Expression is increased for
any enzymes expressed at low levels that can limit the flux through the
pathway by, for example, introduction of additional gene copy numbers.

[0463] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of hexamethylenediamine One
modeling method is the bilevel optimization approach, OptKnock (Burgard
et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to
select gene knockouts that collectively result in better production of
hexamethylenediamine Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA and succinyl-CoA
intermediates of the hexamethylenediamine product. Adaptive evolution is
performed to improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied to
the hexamethylenediamine producer to further increase production.

[0464] For large-scale production of hexamethylenediamine, the above
organism is cultured in a fermenter using a medium known in the art to
support growth of the organism under anaerobic conditions. Fermentations
are performed in either a batch, fed-batch or continuous manner.
Anaerobic conditions are maintained by first sparging the medium with
nitrogen and then sealing the culture vessel, for example, flasks can be
sealed with a septum and crimp-cap. Microaerobic conditions also can be
utilized by providing a small hole in the septum for limited aeration.
The pH of the medium is maintained at a pH of around 7 by addition of an
acid, such as H2SO4. The growth rate is determined by measuring
optical density using a spectrophotometer (600 nm) and the glucose uptake
rate by monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XVII

Preparation of a Caprolactam Producing Microbial Organism Having a Pathway
for Converting Acetyl-CoA and 4-Aminobutyryl-CoA to 6-Aminocaproyl-CoA

[0465] This example describes the generation of a microbial organism
capable of producing caprolactam from acetyl-CoA and 4-aminobutyryl-CoA.

[0466] Escherichia coli is used as a target organism to engineer the
caprolactam pathway shown in FIG. 11 that starts from acetyl-CoA and
4-aminobutyryl-CoA. E. coli provides a good host for generating a
non-naturally occurring microorganism capable of producing caprolactam.
E. coli is amenable to genetic manipulation and is known to be capable of
producing various products, like ethanol, acetic acid, formic acid,
lactic acid, and succinic acid, effectively under anaerobic or
microaerobic conditions.

[0467] To generate an E. coli strain engineered to produce caprolactam,
nucleic acids encoding the requisite enzymes are expressed in E. coli
using well known molecular biology techniques (see, for example,
Sambrook, supra, 2001; Ausubel, supra, 1999). In particular, the paaJ
(NP--415915.1), paaH (NP--415913.1), and maoC
(NP--415905.1) genes encoding the 3-oxo-6-aminohexanoyl-CoA
thiolase, 3-oxo-6-aminohexanoyl-CoA reductase,
3-hydroxy-6-aminohexanoyl-CoA dehydratase activities, respectively, are
cloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under the
PA1/lacO promoter. In addition, the bcd (NP--349317.1) and etfAB
(NP--349315.1 and NP--349316.1) genes encoding
6-aminohex-2-enoyl-CoA reductase activity are cloned into the pZA33
vector (Expressys, Ruelzheim, Germany) under the PA1/lacO promoter.
Lastly, the sucD (NP--904963.1), gabT (NP--417148.1), and cat2
(P38942.2) genes encoding succinyl-CoA reductase (aldehyde forming), GABA
transaminase, and 4-aminobutyryl-CoA/acyl-CoA transferase activities are
cloned into a third compatible plasmid, pZS23, under the PA1/lacO
promoter, to increase the availability of 4-aminobutyryl-CoA. pZS23 is
obtained by replacing the ampicillin resistance module of the pZS13
vector (Expressys, Ruelzheim, Germany) with a kanamycin resistance module
by well-known molecular biology techniques. The three sets of plasmids
are transformed into E. coli strain MG1655 to express the proteins and
enzymes required for caprolactam synthesis.

[0468] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
caprolactam synthesis genes is corroborated using methods well known in
the art for determining polypeptide expression or enzymatic activity,
including for example, Northern blots, PCR amplification of mRNA,
immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
caprolactam is confirmed using HPLC, gas chromatography-mass spectrometry
(GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

[0469] Microbial strains engineered to have a functional caprolactam
synthesis pathway are further augmented by optimization for efficient
utilization of the pathway. Briefly, the engineered strain is assessed to
determine whether any of the exogenous genes are expressed at a rate
limiting level. Expression is increased for any enzymes expressed at low
levels that can limit the flux through the pathway by, for example,
introduction of additional gene copy numbers.

[0470] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of caprolactam. One modeling
method is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of
caprolactam. Adaptive evolution also can be used to generate better
producers of, for example, the acetyl-CoA and succinyl-CoA intermediates
of the caprolactam product. Adaptive evolution is performed to improve
both growth and production characteristics (Fong and Palsson, Nat. Genet.
36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based
on the results, subsequent rounds of modeling, genetic engineering and
adaptive evolution can be applied to the caprolactam producer to further
increase production.

[0471] For large-scale production of caprolactam, the above organism is
cultured in a fermenter using a medium known in the art to support growth
of the organism under anaerobic conditions. Fermentations are performed
in either a batch, fed-batch or continuous manner. Anaerobic conditions
are maintained by first sparging the medium with nitrogen and then
sealing the culture vessel, for example, flasks can be sealed with a
septum and crimp-cap. Microaerobic conditions also can be utilized by
providing a small hole in the septum for limited aeration. The pH of the
medium is maintained at a pH of around 7 by addition of an acid, such as
H2SO4. The growth rate is determined by measuring optical
density using a spectrophotometer (600 nm) and the glucose uptake rate by
monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XVIII

Preparation of a Hexamethylenediamine Producing Microbial Organism Having
a Pathway for Converting Acetyl-CoA and 4-Aminobutyryl-CoA to
6-Aminocaproyl-CoA

[0472] This example describes the generation of a microbial organism
capable of producing hexamethylenediamine from acetyl-CoA and
4-aminobutyryl-CoA.

[0473] Escherichia coli is used as a target organism to engineer the
hexamethylenediamine pathway shown in Figure XVII that starts from
acetyl-CoA and 4-aminobutyryl-CoA. E. coli provides a good host for
generating a non-naturally occurring microorganism capable of producing
hexamethylenediamine E. coli is amenable to genetic manipulation and is
known to be capable of producing various products, like ethanol, acetic
acid, formic acid, lactic acid, and succinic acid, effectively under
anaerobic or microaerobic conditions.

[0475] The resulting genetically engineered organism is cultured in
glucose-containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of the
hexamethylenediamine synthesis genes is corroborated using methods well
known in the art for determining polypeptide expression or enzymatic
activity, including for example, Northern blots, PCR amplification of
mRNA, immunoblotting, and the like. Enzymatic activities of the expressed
enzymes are confirmed using assays specific for the individual
activities. The ability of the engineered E. coli strain to produce
hexamethylenediamine is confirmed using HPLC, gas chromatography-mass
spectrometry (GCMS) and/or liquid chromatography-mass spectrometry
(LCMS).

[0476] Microbial strains engineered to have a functional
hexamethylenediamine synthesis pathway are further augmented by
optimization for efficient utilization of the pathway. Briefly, the
engineered strain is assessed to determine whether any of the exogenous
genes are expressed at a rate limiting level. Expression is increased for
any enzymes expressed at low levels that can limit the flux through the
pathway by, for example, introduction of additional gene copy numbers.

[0477] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of hexamethylenediamine One
modeling method is the bilevel optimization approach, OptKnock (Burgard
et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to
select gene knockouts that collectively result in better production of
hexamethylenediamine Adaptive evolution also can be used to generate
better producers of, for example, the acetyl-CoA and succinyl-CoA
intermediates of the hexamethylenediamine product. Adaptive evolution is
performed to improve both growth and production characteristics (Fong and
Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science
314:1565-1568 (2006)). Based on the results, subsequent rounds of
modeling, genetic engineering and adaptive evolution can be applied to
the hexamethylenediamine producer to further increase production.

[0478] For large-scale production of hexamethylenediamine, the above
organism is cultured in a fermenter using a medium known in the art to
support growth of the organism under anaerobic conditions. Fermentations
are performed in either a batch, fed-batch or continuous manner.
Anaerobic conditions are maintained by first sparging the medium with
nitrogen and then sealing the culture vessel, for example, flasks can be
sealed with a septum and crimp-cap. Microaerobic conditions also can be
utilized by providing a small hole in the septum for limited aeration.
The pH of the medium is maintained at a pH of around 7 by addition of an
acid, such as H2SO4. The growth rate is determined by measuring
optical density using a spectrophotometer (600 nm) and the glucose uptake
rate by monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu, Columbia Md.), for example, using an
Aminex® series of HPLC columns (for example, HPX-87 series) (BioRad,
Hercules Calif.), using a refractive index detector for glucose and
alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.
Bioeng. 775-779 (2005)).

Example XIX

Pathways for Production of 6-Aminocaproic Acid from Succinic Semialdehyde
and Pyruvate

[0479] This example describes exemplary pathways for production of
6-aminocaproic acid.

[0480] Novel pathways for producing 6-aminocaproic acid (6-ACA) and
related products are described herein. These pathways synthesize 6-ACA
from succinic semialdehyde and pyruvate, utilizing aldolase and hydratase
enzymes from the 4-hydroxyphenylacetic acid degradation pathway. The
candidate enzymes, and associated risks of implementation are discussed
in Example XXI below.

[0481] This invention is directed, in part, to non-naturally occurring
microorganisms that express genes encoding enzymes that catalyze 6-ACA
production. Successfully engineering these pathways entails identifying
an appropriate set of enzymes with sufficient activity and specificity,
cloning their corresponding genes into a production host, optimizing the
expression of these genes in the production host, optimizing fermentation
conditions, and assaying for product formation following fermentation.

[0482] 6-aminocaproic acid and derivatives are produced from succinic
semialdehyde and pyruvate in a minimum of five enzymatic steps. In the
first step of all pathways, pyruvate and succinic semialdehyde are joined
by 4-hydroxy-2-oxoheptane-1,7-dioate (HODH) aldolase. The product of this
reaction, HODH, is then dehydrated by 2-oxohept-4-ene-1,7-dioate (OHED)
hydratase to form OHED. In subsequent steps, OHED is transaminated,
decarboxylated or reduced as shown in FIG. 12.

[0483] In one route, the alkene of OHED is reduced by OHED reductase,
forming 2-oxoheptane-1,7-dioate (2-OHD) (FIG. 12, Step C), a 2-ketoacid.
2-OHD is then converted to adipate semialdehyde by a ketoacid
decarboxylase (FIG. 12, Step D). In the final step, the aldehyde of
adipate semialdehyde is converted to an amine by an aminotransferase or
an aminating oxidoreductase (FIG. 12, Step E).

[0484] In a similar route, the 2-keto group of 2-OHD is transaminated by
an aminotransferase or an aminating oxidoreductase (FIG. 12, Step H) to
form 2-aminoheptane-1,7-dioate (2-AHD). This product is then
decarboxylated by 2-AHD decarboxylase to form 6-aminocapropate (FIG. 12,
Step I).

[0485] In an alternate route, OHED is first decarboxylated by OHED
decarboxylase (FIG. 12, Step F), resulting in the formation of
6-oxohex-4-enoate (6-OHE). The alkenal group of 6-OHE is reduced by an
oxidoreductase to adipate semialdehyde (FIG. 12, Step G). Adipate
semialdehyde is then converted to 6-aminocaproate by an aminotransferase
or aminating oxidoreductase (FIG. 12, Step E).

[0486] Yet another route calls for an aminotransferase or aminating
oxidoreductase to convert OHED to 2-aminohept-4-ene-1,7-dioate (2-AHE)
(FIG. 12, Step J). The alkene of 2-AHE is subsequently reduced by an
alkene oxidoreductase (FIG. 12, Step K). The product of this reaction,
2-AHD, is then decarboxylated by an amino acid decarboxylase (FIG. 12,
Step I) to form 6-aminocaproate.

[0487] In yet another route, HODH is converted to 3-hydroxyadipyl-CoA by
either an HODH dehydrogenase or and HODH formate-lyase (FIG. 12, Step L).
3-Hydroxyadipyl-CoA is subsequently dehydrated and reduced to form
adipyl-CoA (FIG. 12, Steps M, N). Adipyl-CoA is reduced and de-acylated
to form adipate semialdehyde (FIG. 12, Step O), which is then converted
to 6-aminocaproate by an aminotransferase or an aminating oxidoreductase
(FIG. 12, Step E).

[0488] In a similar route, HODH is first converted to OHED (FIG. 12, Step
B), as described above. OHED is then converted to 2,3-dehydroadipyl-CoA
by a dehydrogenase or an OHED formate-lyase (FIG. 12, Step P).
2,3-Dihydroadipyl-CoA is then reduced to adipyl-CoA (FIG. 12, Step N),
which is converted to 6-aminocaproate via adipate semialdehyde (FIG. 12,
Steps O, E), as described previously.

[0489] In the final route, HODH is converted to 2-OHD via steps B and C,
as described previously. A 2-OHD formate-lyase or dehydrogenase converts
2-OHD to adipyl-CoA (FIG. 12, Step Q), which is then reduced by a
CoA-dependent aldehyde dehydrogenase (FIG. 12, Step O). The product,
adipate semialdehyde, is converted to 6-aminocaproate by an
aminotransferase or aminating oxidoreductase (FIG. 12, Step E).

[0490] The routes detailed in FIG. 12 are able to achieve the maximum
theoretical 6-ACA yield of 0.8 moles 6-ACA per mole glucose utilized. The
energetic yield is also favorable, with a maximum of 1.6 moles ATP per
mole glucose utilized at the maximum product yield. The following
assumptions were used to calculate yield: 1) phosphoenolpyruvate (PEP)
carboxykinase is able to operate in the ATP-generating direction, 2) NH4
and 6-ACA are transported into the cell by proton antiport, and 3)
succinic semialdehyde is formed from alpha-ketoglutarate and/or
succinyl-CoA. Succinic semialdehyde dehydrogenase is a NAD(P)H and
CoA-dependent aldehyde dehydrogenase that converts succinyl-CoA to
succinic semialdehyde. Succinic semialdehyde is formed from
alpha-ketoglutarate by two enzymes: alpha-ketoglutarate decarboxylase and
4-aminobutyrate transaminase.

Example XX

Pathways for Production of Hexamethylenediamine from 6-Aminocaproate

[0491] This example describes exemplary pathways for production of
hexamethylenediamine

[0492] Novel pathways for producing hexamethylenediamine (HMDA) and
related products are described herein. This pathway synthesizes HMDA from
6-Aminocaproate (6-ACA). These pathways involve activation of the acid
group by phosphorylation and/or acylation. Acetylation of the terminal
amino group provides protection from spontaneous cyclization of pathway
intermediates. The candidate enzymes, and associated risks of
implementation are discussed in Example XXI below.

[0493] This invention is directed, in part, to non-naturally occurring
microorganisms that express genes encoding enzymes that catalyze HMDA
production. Successfully engineering these pathways entails identifying
an appropriate set of enzymes with sufficient activity and specificity,
cloning their corresponding genes into a production host, optimizing the
expression of these genes in the production host, optimizing fermentation
conditions, and assaying for product formation following fermentation.

[0494] Several pathways for producing HMDA from 6-aminocaproate are
detailed in FIG. 13. All routes entail activation of the carboxylic acid
group, followed by reduction and transamination. In three routes,
6-aminocaproate is activated directly while in other routes, the terminal
amine group is protected by N-acetylation to prevent spontaneous
cyclization.

[0495] In one route, 6-aminocaproate is phosphorylated to 6-AHOP by
6-aminocaproate kinase (FIG. 13, Step A). 6-AHOP is then reduced to
6-aminocaproic semialdehyde (FIG. 13, Step B) and subsequently
transaminated (FIG. 13, Step C) by an aminotransferase or an aminating
oxidoreductase.

[0496] Alternately, 6-AHOP is converted to 6-aminocaproyl-CoA by an
acyltransferase (FIG. 13, Step L). 6-Aminocaproyl-CoA is then reduced to
6-aminocaproic semialdehyde by a CoA-dependent aldehyde dehydrogenase
(FIG. 13, Step N). HMDA is then formed by transamination of
6-aminocaproic semialdehyde by an aminotransferase or aminating
oxidoreductase (FIG. 13, Step C).

[0497] In yet another route, 6-aminocaproate is first activated to a CoA
derivative by a CoA transferase or CoA ligase (FIG. 13, Step M). The
product, 6-aminocaproyl-CoA, may spontaneously cyclize, or be converted
to 6-aminocaproic semialdehyde by an aldehyde-forming CoA-dependent
aldehyde dehydrogenase (FIG. 13, Step N). 6-Aminocaproic semialdehyde is
converted to HMDA by an aminotransferase or an aminating oxidoreductase
(FIG. 13, Step C).

[0498] Additional routes proceed from 6-acetamidohexanoate, the acetylated
product of 6-aminocaproate N-acetyltransferase. 6-Acetamidohexanoate is
converted to 6-acetamidohexanal by different routes (described below). In
the final two steps of these routes, 6-acetamidohexanal is first
converted to 6-acetamidohexanamine by an aminotransferase or an aminating
oxidoreductase (FIG. 13, Step G). 6-Acetamidohexanamine is subsequently
converted to HMDA by an amide hydrolase or an N-acetyltransferase (FIG.
13, Step H).

[0499] In one route, 6-acetamidohexanoate is phosphorylated by
6-acetamidohexanoate kinase (FIG. 13, Step E). The product, 6-AAHOP, is
reduced to form 6-acetamidohexanal (FIG. 13, Step F), which is then
converted to HMDA as described above.

[0500] In another route, 6-acetamidohexanoate is activated to
6-acetamidohexanoyl-CoA by a CoA transferase or CoA ligase (FIG. 13, Step
I). The CoA derivative is then reduced to 6-acetamidohexanal by an
aldehyde-forming CoA-dependent oxidoreductase (FIG. 13, Step J).
6-acetamidohexanal is then converted to HMDA as described above.

[0501] Alternately, 6-acetamidohexanoate is phosphorylated to 6-AAHOP
(FIG. 13, Step E) and subsequently converted to 6-acetamidohexanoyl-CoA
by an acyltransferase (FIG. 13, Step K). 6-Acetamidohexanoyl-CoA is then
reduced to HMDA as described previously.

Example XXI

Enzyme Classification System for Production of 6-Aminocaproic Acid and
Hexamethylenediamine

[0502] This example describes the enzyme classification system for the
exemplary pathways described in Examples XIX and XX for production of
6-aminocaproate or hexamethylenediamine

[0503] All transformations depicted in FIGS. 12 and 13 fall into the
general categories of transformations shown in Table 9. Below is
described a number of biochemically characterized genes in each category.
Specifically listed are genes that can be applied to catalyze the
appropriate transformations in FIGS. 12-13 when properly cloned and
expressed.

[0504] Table 9 shows the enzyme types useful to convert common central
metabolic intermediates into 6-aminocaproate and hexamethylenediamine.
The first three digits of each label correspond to the first three Enzyme
Commission number digits which denote the general type of transformation
independent of substrate specificity.

[0507] An additional enzyme that converts an acyl-CoA to its corresponding
aldehyde is malonyl-CoA reductase which transforms malonyl-CoA to malonic
semialdehyde. Malonyl-CoA reductase is a key enzyme in autotrophic carbon
fixation via the 3-hydroxypropionate cycle in thermoacidophilic archaeal
bacteria (Berg et al., Science 318:1782-1786 (2007); and Thauer, R. K.,
Science. 318:1732-1733 (2007)). The enzyme utilizes NADPH as a cofactor
and has been characterized in Metallosphaera and Sulfolobus sp (Alber et
al., J. Bacteriol. 188:8551-8559 (2006); and Hugler et al., J. Bacteriol.
184:2404-2410 (2002)). The enzyme is encoded by Msed--0709 in
Metallosphaera sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006);
and Berg et al., Science. 318:1782-1786 (2007)). A gene encoding a
malonyl-CoA reductase from Sulfolobus tokodaii was cloned and
heterologously expressed in E. coli (Alber et al., J. Bacteriol.
188:8551-8559 (2006)). This enzyme has also been shown to catalyze the
conversion of methylmalonyl-CoA to its corresponding aldehyde (WIPO
Patent Application WO/2007/141208 Kind Code: A2). Although the aldehyde
dehydrogenase functionality of these enzymes is similar to the
bifunctional dehydrogenase from Chloroflexus aurantiacus, there is little
sequence similarity. Both malonyl-CoA reductase enzyme candidates have
high sequence similarity to aspartate-semialdehyde dehydrogenase, an
enzyme catalyzing the reduction and concurrent dephosphorylation of
aspartyl-4-phosphate to aspartate semialdehyde. Additional gene
candidates can be found by sequence homology to proteins in other
organisms including Sulfolobus solfataricus and Sulfolobus acidocaldarius
and have been listed below. Yet another candidate for CoA-acylating
aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii
(Toth et al., Appl Environ Microbiol 65:4973-4980 (1999)). This enzyme
has been reported to reduce acetyl-CoA and butyryl-CoA to their
corresponding aldehydes. This gene is very similar to eutE that encodes
acetaldehyde dehydrogenase of Salmonella typhimurium and E. coli (Toth et
al., Appl Environ Microbiol 65:4973-4980 (1999)).

[0509] Several transformations in FIG. 12 require conversion of a
2-ketoacid to an acyl-CoA (Steps L, P and Q) by an enzyme in the EC class
1.2.1. Such reactions are catalyzed by multi-enzyme complexes that
catalyze a series of partial reactions which result in acylating
oxidative decarboxylation of 2-keto-acids. Exemplary enzymes include 1)
branched-chain 2-keto-acid dehydrogenase, 2) alpha-ketoglutarate
dehydrogenase, and 3) the pyruvate dehydrogenase multienzyme complex
(PDHC). Each of the 2-keto-acid dehydrogenase complexes occupies key
positions in intermediary metabolism, and enzyme activity is typically
tightly regulated (Fries et al., Biochemistry 42:6996-7002 (2003)). The
enzymes share a complex but common structure composed of multiple copies
of three catalytic components: alpha-ketoacid decarboxylase (E1),
dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase
(E3). The E3 component is shared among all 2-keto-acid dehydrogenase
complexes in an organism, while the E1 and E2 components are encoded by
different genes. The enzyme components are present in numerous copies in
the complex and utilize multiple cofactors to catalyze a directed
sequence of reactions via substrate channeling. The overall size of these
dehydrogenase complexes is very large, with molecular masses between 4
and 10 million Da (i.e. larger than a ribosome).

[0510] Activity of enzymes in the 2-keto-acid dehydrogenase family is
normally low or limited under anaerobic conditions in E. coli. Increased
production of NADH (or NADPH) could lead to a redox-imbalance, and NADH
itself serves as an inhibitor to enzyme function. Engineering efforts
have increased the anaerobic activity of the E. coli pyruvate
dehydrogenase complex (Kim et al., Appl. Environ. Microbiol. 73:1766-1771
(2007); Kim et al., J. Bacteriol. 190:3851-3858 (2008); and Zhou et al.,
Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effect
of NADH can be overcome by engineering an H322Y mutation in the E3
component (Kim et al., J. Bacteriol. 190:3851-3858 (2008)). Structural
studies of individual components and how they work together in complex
provide insight into the catalytic mechanisms and architecture of enzymes
in this family (Aevarsson et al., Nat. Struct. Biol. 6:785-792 (1999);
and Zhou et al., Proc. Natl. Acad. Sci. U.S. A 98:14802-14807 (2001)).
The substrate specificity of the dehydrogenase complexes varies in
different organisms, but generally branched-chain keto-acid
dehydrogenases have the broadest substrate range.

[0511] Alpha-ketoglutarate dehydrogenase (AKGD) converts
alpha-ketoglutarate to succinyl-CoA and is the primary site of control of
metabolic flux through the TCA cycle (Hansford, Curr. Top. Bioenerg.
10:217-278 (1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD
gene expression is downregulated under anaerobic conditions and during
growth on glucose (Park et al., Mol. Microbiol. 15:473-482 (1995)).
Although the substrate range of AKGD is narrow, structural studies of the
catalytic core of the E2 component pinpoint specific residues responsible
for substrate specificity (Knapp et al., J. Mol. Biol. 280:655-668
(1998)). The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and
pdhD (E3, shared domain), is regulated at the transcriptional level and
is dependent on the carbon source and growth phase of the organism
(Resnekov et al., Mol. Gen. Genet. 234:285-296 (1992)). In yeast, the
LPD1 gene encoding the E3 component is regulated at the transcriptional
level by glucose (Roy and Dawes, J. Gen. Microbiol. 133:925-933 (1987)).
The E1 component, encoded by KGD1, is also regulated by glucose and
activated by the products of HAP2 and HAP3 (Repetto and Tzagoloff, Mol.
Cell. Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited by
products NADH and succinyl-CoA, is well-studied in mammalian systems, as
impaired function of has been linked to several neurological diseases
(Tretter and dam-Vizi, Philos. Trans. R. Soc. Lond B Biol. Sci.
360:2335-2345 (2005)).

[0514] As an alternative to the large multienzyme 2-keto-acid
dehydrogenase complexes described above, some anaerobic organisms utilize
enzymes in the 2-ketoacid oxidoreductase family (OFOR) to catalyze
acylating oxidative decarboxylation of 2-keto-acids. Unlike the
dehydrogenase complexes, these enzymes contain iron-sulfur clusters,
utilize different cofactors, and use ferredoxin or flavodoxin as electron
acceptors in lieu of NAD(P)H. While most enzymes in this family are
specific to pyruvate as a substrate (POR) some 2-keto-acid:ferredoxin
oxidoreductases have been shown to accept a broad range of 2-ketoacids as
substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukuda and
Wakagi, Biochim. Biophys. Acta 1597:74-80 (2002); and Zhang et al., J.
Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from the
thermoacidophilic archaeon Sulfolobus tokodaii 7, which contains an alpha
and beta subunit encoded by gene ST2300 (Fukuda and Wakagi, Biochim.
Biophys. Acta 1597:74-80 (2002); and Zhang et al., J. Biochem.
120:587-599 (1996)). A plasmid-based expression system has been developed
for efficiently expressing this protein in E. coli (Fukuda et al., Eur.
J. Biochem. 268:5639-5646 (2001)) and residues involved in substrate
specificity were determined (Fukuda and Wakagi, Biochim. Biophys. Acta
1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str. K1 have also
been recently cloned into E. coli, characterized, and found to react with
a broad range of 2-oxoacids (Nishizawa et al., FEBS Lett. 579:2319-2322
(2005)). The gene sequences of these OFOR candidates are available,
although they do not have GenBank identifiers assigned to date. There is
bioinformatic evidence that similar enzymes are present in all archaea,
some anaerobic bacteria and amitochondrial eukarya (Fukuda and Wakagi,
Biochim. Biophys. Acta 1597:74-80 (2002)). This class of enzyme is also
interesting from an energetic standpoint, as reduced ferredoxin could be
used to generate NADH by ferredoxin-NAD reductase (Petitdemange et al.,
Biochim. Biophys. Acta 421:334-337 (1976)). Also, since most of the
enzymes are designed to operate under anaerobic conditions, less enzyme
engineering may be required relative to enzymes in the 2-keto-acid
dehydrogenase complex family for activity in an anaerobic environment.

[0518] Several transformations fall into the category of oxidoreductases
that reduce an alkene to an alkane (EC 1.3.1.-). For example, Steps C, G,
K and N in FIG. 12, catalyzed by OHED reductase, 6-OHE reductase, 2-AHE
reductase and 2,3-dehydroadipyl-CoA reductase, respectively, fall into
this category. Enone reductase, alkenal reductase, and enoate reductase
enzymes are suitable enzyme candidates for catalyzing the transformations
of Steps C, G and K. Enoyl-CoA reductase enzymes catalyze the conversion
of 2,3-dehydroadipyl-CoA to adipyl-CoA (Step N).

[0519] Enzymes with enone reductase activity have been identified in
prokaryotes, eukaryotes and plants (Shimoda et al., Bulletin of the
chemical Society of Japan 77:2269-2 (2004); and Wanner and Tressl, Eur.
J. Biochem. 255:271-278 (1998)). Two enone reductases from the cytosolic
fraction of Saccharomyces cerevisiae were purified and characterized, and
found to accept a variety of alkenals (similar to 6-OHE) and enoyl
ketones (similar to OHED) as substrates (Wanner and Tressl, Eur. J.
Biochem. 255:271-278 (1998)). Genes encoding these enzymes have not been
identified to date. Cell extracts of cyanobacterium Synechococcus sp.
PCC7942 reduced a variety enone substrates to their corresponding alkyl
ketones (Shimoda et al., Bulletin of the chemical Society of Japan
77:2269-2 (2004)). Genes have not been associated with this activity in
this organism. Enone reductases in other organisms can also catalyze this
transformation.

[0522] 2-Enoate reductase enzymes are known to catalyze the
NAD(P)H-dependent reduction of a wide variety of α,
β-unsaturated carboxylic acids and aldehydes (Rohdich et al., J.
Biol. Chem. 276:5779-5787 (2001)). In the recently published genome
sequence of C. kluyveri, 9 coding sequences for enoate reductases were
reported, out of which one has been characterized (Seedorf et al., Proc.
Natl. Acad. Sci. U.S. A 105:2128-2133 (2008)). The enr genes from both C.
tyrobutyricum and M. thermoaceticum have been cloned and sequenced and
show 59% identity to each other. The former gene is also found to have
approximately 75% similarity to the characterized gene in C. kluyveri
(Giesel and Simon, Arch. Microbiol 135:51-57 (1983)). It has been
reported based on these sequence results that enr is very similar to the
dienoyl CoA reductase in E. coli (fadH) (Rohdich et al., J. Biol. Chem.
276:5779-5787 (2001)). The C. thermoaceticum enr gene has also been
expressed in a catalytically active form in E. coli (Rohdich et al., J.
Biol. Chem. 276:5779-5787 (2001)).

[0524] Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the
reduction of 2,3-dehydroadipyl-CoA to adipyl-CoA (FIG. 12, Step N). One
exemplary enoyl-CoA reductase is the gene product of bcd from C.
acetobutylicum (Atsumi et al., Metab Eng 10:305-311 (2008); and Boynton
et al., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes
the reduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can
be enhanced by expressing bcd in conjunction with expression of the C.
acetobutylicum etfAB genes, which encode an electron transfer
flavoprotein. An additional candidate for the enoyl-CoA reductase step is
the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeister et
al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived from this
sequence following the removal of its mitochondrial targeting leader
sequence was cloned in E. coli resulting in an active enzyme (Hoffmeister
et al., J. Biol. Chem. 280:4329-4338 (2005)). This approach is well known
to those skilled in the art of expressing eukaryotic genes, particularly
those with leader sequences that may target the gene product to a
specific intracellular compartment, in prokaryotic organisms. A close
homolog of this gene, TDE0597, from the prokaryote Treponema denticola
represents a third enoyl-CoA reductase which has been cloned and
expressed in E. coli (Tucci and Martin, Febs Letters 581:1561-1566
(2007)).

[0526] An additional candidate is 2-methyl-branched chain enoyl-CoA
reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically
hindered trans-enoyl-CoA substrates. This enzyme participates in
branched-chain fatty acid synthesis in the nematode Ascarius suum and is
capable of reducing a variety of linear and branched chain substrates
including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and
pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993)). Two
isoforms of the enzyme, encoded by genes acad1 and acad, have been
characterized.

[0528] Oxidoreductases in the EC class 1.4.1 that convert an aldehyde or
ketone to its corresponding amine group catalyze several biosynthetic
steps in the disclosed pathways. In FIG. 12, the conversions of OHED to
2-AHE (Step J), 2-OHD to 2-AHD (Step H) and adipate semialdehyde to
6-aminocaproate (Step E) are catalyzed by OHED aminating oxidoreductase,
2-OHD aminating oxidoreductase and adipate semialdehyde aminating
oxidoreductase. In FIG. 13, conversion of 6-aminocaproate semialdehyde to
HMDA (Step H) and 6-acetamidohexanal to 6-acetamidohexanamine (Step G),
are also catalyzed by aminating oxidoreductases.

[0535] One candidate enzyme for acetylating 6-ACA is lysine
N-acetyltransferase (EC 2.3.1.32), an enzyme which selectively transfers
the acetyl moiety from acetyl phosphate to the terminal amino group of
L-lysine, beta-L-lysine or L-ornithine. Although this enzyme is not known
to acetylate 6-ACA, this substrate is structurally similar to the natural
substrate. Lysine N-acetyltransferase has been characterized in Bos
taurus (Paik. and Kim, Arch. Biochem. Biophys. 108:221-229, 1964) and
Methanosarcina mazei (Pfluger et al., Appl Environ. Microbiol.
69:6047-6055 (2003)). Methanogenic archaea M. maripaludis, M.
acetivorans, M. barkeri and M. jannaschii are also predicted to encode
enzymes with this functionality (Pfluger et al., Appl Environ. Microbiol.
69:6047-6055 (2003)).

[0542] Additional enzyme candidates include putrescine aminotransferases
or other diamine aminotransferases. Such enzymes are particularly well
suited for carrying out the conversion of 6-aminocaproate semialdehyde to
HMDA. The E. coli putrescine aminotransferase is encoded by the ygjG gene
and the purified enzyme also was able to transaminate cadaverine and
spermidine (Samsonova et al., BMC. Microbiol. 3:2 (2003)). In addition,
activity of this enzyme on 1,7-diaminoheptane and with amino acceptors
other than 2-oxoglutarate (e.g., pyruvate, 2-oxobutanoate) has been
reported (Kim, J Biol. Chem. 239:783-786 (1964); and Samsonova et al.,
BMC. Microbiol. 3:2 (2003)). A putrescine aminotransferase with higher
activity with pyruvate as the amino acceptor than alpha-ketoglutarate is
the spuC gene of Pseudomonas aeruginosa (Lu et al., J. Bacteriol.
184:3765-3773 (2002)).

[0544] Steps J and H of FIG. 12 are catalyzed by aminotransferases that
transform amino acids into oxo-acids. In Step J, OHED is transaminated to
form 2-AHE by OHED aminotransferase. The transamination of 2-OHD to 2-AHD
by 2-OHD aminotransferase (Step H) is a similar reaction. An exemplary
enzyme candidate for catalyzing these reactions is aspartate
aminotransferase, an enzyme that naturally transfers an oxo group from
oxaloacetate to glutamate, forming alpha-ketoglutarate and aspartate.
Aspartate is similar in structure to OHED and 2-AHD. Aspartate
aminotransferase activity is catalyzed by, for example, the gene products
of aspC from Escherichia coli (Yagi et al., FEBS Lett. 100:81-84, (1979);
and Yagi et al., Methods Enzymol. 113:83-89 (1985)), AAT2 from
Saccharomyces cerevisiae (Yagi et al., J. Biochem. 92:35-43 (1982)) and
ASPS from Arabidopsis thaliana (de la Torre et al., Plant J 46:414-425
(2006); Kwok and Hanson, J Exp. Bot. 55:595-604 (2004); and Wilkie and
Warren, Protein Expr. Purif. 12:381-389 (1998)). The enzyme from Rattus
norvegicus has been shown to transaminate alternate substrates such as
2-aminohexanedioic acid and 2,4-diaminobutyric acid (Recasens et al.,
Biochemistry 19:4583-4589 (1980)) Aminotransferases that work on other
amino-acid substrates can catalyze this transformation. Valine
aminotransferase catalyzes the conversion of valine and pyruvate to
2-ketoisovalerate and alanine The E. coli gene, avtA, encodes one such
enzyme (Whalen and Berg, C. J. Bacteriol. 150:739-746 (1982)). This gene
product also catalyzes the transamination of α-ketobutyrate to
generate α-aminobutyrate, although the amine donor in this reaction
has not been identified (Whalen and Berg, J. Bacteriol. 158:571-574
(1984)). The gene product of the E. coli serC catalyzes two reactions,
phosphoserine aminotransferase and phosphohydroxythreonine
aminotransferase (Lam and Winkler, J. Bacteriol. 172:6518-6528 (1990)),
and activity on non-phosphorylated substrates could not be detected
(Drewke et al., FEBS. Lett. 390:179-182 (1996)).

[0549] Coenzyme-A (CoA) transferases catalyze the reversible transfer of a
CoA moiety from one molecule to another. In Step M of FIG. 13,
3-aminocaproyl-CoA is formed by the transfer of a CoA group from
acetyl-CoA, succinyl-CoA, or another CoA donor. A similar transformation
is catalyzed by 6-acetamidohexanoate CoA-transferase, shown in Step I of
FIG. 13. Exemplary CoA transferase candidates are catalyzed by the gene
products of cat1, cat2, and cat3 of Clostridium kluyveri which have been
shown to exhibit succinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA
transferase activity, respectively (Seedorf et al., Proc. Natl. Acad.
Sci. U.S. A 105:2128-2133 (2008); and Sohling and Gottschalk, J.
Bacteriol. 178:871-880 (1996)). Similar CoA transferase activities are
also present in Trichomonas vaginalis (van Grinsven et al., J. Biol.
Chem. 283:1411-1418 (2008)) and Trypanosoma brucei (Riviere et al., J.
Biol. Chem. 279:45337-45346 (2004)).

[0554] Deacetylation of linear acetamides is catalyzed by an
amidohydrolase in the 3.5.1 family of enzymes. Such an enzyme is required
for the deacetylation of 6-acetamidohexanamine to HMDA (FIG. 13, Step H).
An enzyme catalyzing a similar transformation is 4-acetamidobutyrate
deacetylase (EC 3.5.1.63), which naturally deacetylates
4-acetamidobutyrate. The enzyme, studied for its role in putrescine
degradation in Candida boidinii (Gillyon et al., Journal of General
Microbiology 133:2477-2485 (1987)), has been shown to deacetylate a
variety of substrates including 6-acetamidohexanoate (Haywood and Large,
Journal of General Microbiology 132:7-14 (1986)). Although
6-Acetamidohexanoate is similar in structure to the desired substrate,
deacetylation of this compound (FIG. 13, step D, reverse reaction) may
hinder efficient production of HMDA. Protein engineering or directed
evolution may be required to improve specificity for
6-acetamidohexanamine. The gene associated with this activity has not
been identified to date.

[0555] Acetylpolyamine amidohydrolase (EC 3.5.1.62), is another candidate
enzyme that forms the diamines putrescine and cadaverine from their
acetylated precursors. The acetylpolyamine deacetylase (AphA) from
Mycoplana ramosa has been cloned in E. coli and characterized (Sakurada
et al., J. Bacteriol. 178:5781-5786 (1996)) and a crystal structure is
available (Fujishiro et al., Biochem. Biophys. Res. Commun. 157:1169-1174
(1988)). This enzyme has also been studied in Micrococcus luteus, but the
associated gene has not been identified to date (Suzuki et al., Biochim.
Biophys. Acta 882:140-142 (1986)). A protein the histone deacetylase
superfamily with high sequence similarity to AphA was identified in the
M. luteus genome (evalue=1e-18, 37% identity). The N-acetyl-L-ornithine
deacetylase from E. coli is another candidate amidohydrolase (EC
3.5.1.16). The E. coli enzyme, encoded by the argE gene (McGregor et al.,
J Am. Chem. Soc. 127:14100-14107 (2005); and Meinnel et al., J.
Bacteriol. 174:2323-2331 (1992)), removes N-acetyl groups from a variety
of substrates including ornithine, lysine, glutamine, and other amino
acids (Javid-Majd and Blanchard, Biochemistry 39:1285-1293 (2000)).

[0558] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad
substrate range and has been the target of enzyme engineering studies.
The enzyme from Pseudomonas putida has been extensively studied and
crystal structures of this enzyme are available (Hasson et al.,
Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry
42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the
active site of the Pseudomonas putida enzyme altered the affinity (Km) of
naturally and non-naturally occurring substrates (Siegert et al., Protein
Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been
further modified by directed engineering (Lingen et al., Protein Eng
15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The
enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been
characterized experimentally (Barrowman et al., FEMS Microbiology Letters
34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri,
Pseudomonas fluorescens and other organisms can be inferred by sequence
homology or identified using a growth selection system developed in
Pseudomonas putida (Henning et al., Appl. Environ. Microbiol.
72:7510-7517 (2006)).

[0559] A third enzyme capable of decarboxylating 2-oxoacids is
alpha-ketoglutarate decarboxylase (KGD). The substrate range of this
class of enzymes has not been studied to date. The KDC from Mycobacterium
tuberculosis (Tian et al., Proc Natl Acad Sci U S. A 102:10670-10675
(2005)) has been cloned and functionally expressed in other internal
projects at Genomatica. However, it is not an ideal candidate for strain
engineering because it is large (˜130 kD) and GC-rich. KDC enzyme
activity has been detected in several species of rhizobia including
Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J.
Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have
not been isolated in these organisms, the genome sequences are available
and several genes in each genome are annotated as putative KDCs. A KDC
from Euglena gracilis has also been characterized but the gene associated
with this activity has not been identified to date (Shigeoka and Nakano,
Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids
starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID
NO: 1) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)).
The gene can be identified by testing candidate genes containing this
N-terminal sequence for KDC activity.

[0560] A fourth candidate enzyme for catalyzing this step is branched
chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been
shown to act on a variety of compounds varying in chain length from 3 to
6 carbons (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988); and Smit
et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme in
Lactococcus lactis has been characterized on a variety of branched and
linear substrates including 2-oxobutanoate, 2-oxohexanoate,
2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and
isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311 (2005)). The
enzyme has been structurally characterized (Berg et al., Science.
318:1782-1786 (2007)). Sequence alignments between the Lactococcus lactis
enzyme and the pyruvate decarboxylase of Zymomonas mobilus indicate that
the catalytic and substrate recognition residues are nearly identical
(Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme
would be a promising candidate for directed engineering. Decarboxylation
of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis;
however, this activity was low (5%) relative to activity on other
branched-chain substrates (Oku and Kaneda, J Biol. Chem. 263:18386-18396
(1988)) and the gene encoding this enzyme has not been identified to
date. Additional BCKA gene candidates can be identified by homology to
the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp
hits to this enzyme are annotated as indolepyruvate decarboxylases (EC
4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that
catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in
plants and plant bacteria.

[0571] Steps I and M of FIG. 13 require acid-thiol ligase or CoA
synthetase functionality to transform 6-ACA and 6-acetamidohexanoate into
their corresponding CoA derivatives (the terms ligase, synthetase, and
synthase are used herein interchangeably and refer to the same enzyme
class). Enzymes catalyzing these exact transformations have not been
characterized to date; however, several enzymes with broad substrate
specificities have been described in the literature. ADP-forming
acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme that couples the
conversion of acyl-CoA esters to their corresponding acids with the
concomitant synthesis of ATP. ACD I from Archaeoglobus fulgidus, encoded
by AF1211, was shown to operate on a variety of linear and branched-chain
substrates including isobutyrate, isopentanoate, and fumarate (Musfeldt
and Schonheit, J. Bacteriol. 184:636-644 (2002)). A second reversible ACD
in Archaeoglobus fulgidus, encoded by AF1983, was also shown to have a
broad substrate range with high activity on cyclic compounds
phenylacetate and indoleacetate (Musfeldt and Schonheit, J. Bacteriol.
184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as
a succinyl-CoA synthetase) accepts propionate, butyrate, and
branched-chain acids (isovalerate and isobutyrate) as substrates, and was
shown to operate in the forward and reverse directions (Brasen and
Schonheit, Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by
PAE3250 from hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed
the broadest substrate range of all characterized ACDs, reacting with
acetyl-CoA, isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA
(Brasen and Schonheit, Arch. Microbiol. 182:277-287 (2004)). Directed
evolution or engineering can be used to modify this enzyme to operate at
the physiological temperature of the host organism. The enzymes from A.
fulgidus, H. marismortui and P. aerophilum have all been cloned,
functionally expressed, and characterized in E. coli (Brasen and
Schonheit, Arch. Microbiol. 182:277-287 (2004); and Musfeldt and
Schonheit, J. Bacteriol. 184:636-644 (2002)). An additional candidate is
the enzyme encoded by sucCD in E. coli, which naturally catalyzes the
formation of succinyl-CoA from succinate with the concomitant consumption
of one ATP, a reaction which is reversible in vivo (Buck et al.,
Biochemistry 24:6245-6252 (1985)).

[0574] E. coli was assayed for tolerance, metabolic activity and growth
during exposure to various concentrations of 6-aminocaproate (6-ACA).
Aerobically, cultures were able to grow media with up to 10% 6-ACA, while
anaerobic cultures could grow in media with approximately 6% 6-ACA (FIG.
15). Because the pathway for producing 6-ACA could require anaerobic
conditions, all other further testing was performed under anaerobic
conditions. To assay tolerance, cultures were grown anaerobically to
mid-log (0.3 OD) and early stationary phase (0.6 OD), the cells were spun
down and resuspended in medium containing various concentrations of
6-ACA. The cultures were grown in capped microfuge tubes, grown overnight
and the ODs of the cultures were assayed (FIG. 16). Under these
conditions, cultures were able to grow (double at least 1 time) in up to
10% 6-ACA. The additional tolerance could have been from the additional
glucose from resuspending the cultures in fresh M9-glucose medium or from
limited oxygen that was present in the capped microfuge tube. To
determine if the cells were metabolically active in the presence of
6-ACA, samples were taken and assayed for ethanol production (FIG. 17).
Ethanol production (and thus metabolic activity) closely tracked with OD
suggesting that if cells are present, they are likely to be metabolically
active. This is helpful to understand because it suggests that even
though cells may be growth inhibited by the accumulation of a product,
they can still continue to produce product.

[0575] At high concentrations (>65 g/L) the osmolarity of 6-ACA is
˜0.5 M which may cause osmotic stress. To determine osmotic stress
as the basis for 6-ACA growth inhibition, cultures were grown in various
concentrations of 6-ACA with and without the osmoprotectant glycine
betaine. As seen in FIG. 18, anaerobic growth in medium with up to 10-12%
6-ACA can be achieved if glycine betaine is present but only 4-6% without
glycine betaine. Therefore much of the toxicity of 6-ACA is likely due to
the osmotic stress. However, it should be noted that 6-ACA is similar to
the amino acid lysine and could have a greater toxic effect in the cell
cytoplasm vs. outside the cell.

Example XXIII

Demonstration of Enzyme Activity for Condensing Succinyl-CoA and
Acetyl-CoA to Form β-ketoadipyl-CoA

[0577] The genes were expressed in E. coli and the proteins purified using
Ni-NTA spin columns and quantified. To assay enzyme activity in vitro, a
5× CoA:DTNB (Ellman's reagent or 5,5'-dithiobis-(2-nitrobenzoic
acid)) mixture was prepared. The mixture consisted of 10 mM succinyl-CoA,
5 mM acetyl-CoA, 30 mM DTNB in 100 mM Tris buffer, pH 7.4. Five μL of
the CoA:DTNB mixture was added to 0.5 μM purified thiolase enzyme in
100 mM Tris buffer, pH 7.8 in a final volume of 50 μL. The reaction
was incubated at 30° C. for 30 minutes, then quenched with 2.5
μL 10% formic acid and samples frozen at -20° C. until ready
for analysis by LC/MS. Because many thiolases can condense two acetyl-CoA
molecules into acetoaceytl-CoA, production of acetoacetyl-CoA was
examined. FIG. 19 shows that 3 thiolases demonstrated thiolase activity
which resulted in acetoacetyl-CoA formation. These were fadAx from
Pseudomonas putida, thiA from Clostridium acetobutylicum and thiB also
from Clostridium acetobutylicum. When enzyme assays were examined for
condensation of succinyl-CoA and acetyl-CoA into β-ketoadipyl-CoA,
several candidates demonstrated the desired activity; paaJ from
Escherichia coli (Nogales et al., Microbiol. 153:357-365 (2007)), phaD
from Pseudomonas putida (Olivera et al., Proc. Natl. Acad. Sci. USA
95:6419-6424 (1998)), bkt from Burkholderia ambifaria AMMD, pcaF from
Pseudomonas putida KT2440 (Harwood et al., J. Bacteriol. 176:6479-6488
(1994)), and pcaF from Pseudomonas aeruginosa PAO1. There was excellent
specificity between the thiolases. Those that generated significant
amounts of β-ketoadipyl-CoA did not produce significant amounts of
acetoacetyl-CoA and likewise those that made acetoacetyl-CoA did not make
detectable amounts of β-ketoadipyl-CoA.

Example XXIV

Pathways for Production of Hexamethylenediamine from Glutamate,
Glutaryl-CoA or Pyruvate and 4-Aminobutanal

[0578] This example describes exemplary pathways for production of
hexamethylenediamine (HMDA) from glutamate, glutaryl-CoA, pyruvate and
4-aminobutanal, or 2-amino-7-oxosubarate through homolysine, the
seven-carbon analog of lysine. Homolysine is an attractive precursor to
HMDA. Although homolysine is a potentially valuable precursor, it is not
a known metabolic intermediate of any organism. Homolysine can be formed
biocatalytically from the central metabolic precursors glutamate,
glutaryl-CoA or pyruvate and 4-aminobutanal. Subsequent decarboxylation
of homolysine by an enzyme analogous to lysine decarboxylase yields HMDA.

[0579] This example describes additional pathways that proceed from
2-amino-7-oxosubarate, or pyruvate and 4-aminobutanal through the
intermediate 6-aminohexanal. 6-Aminohexanal can readily be converted to
HMDA by an aminotransferase or an aminating oxidoreductase.

[0581] Novel pathways for producing hexamethylenediamine (HMDA) and
related products are described herein. The candidate enzymes, and
associated risks of implementation are discussed in Example XXVI below.

[0582] This invention is directed, in part, to non-naturally occurring
microorganisms that express genes encoding enzymes that catalyze HMDA
production. Successfully engineering these pathways entails identifying
an appropriate set of enzymes with sufficient activity and specificity,
cloning their corresponding genes into a production host, optimizing the
expression of these genes in the production host, optimizing fermentation
conditions, and assaying for product formation following fermentation.

[0583] HMDA can be produced from glutamate via glutaryl-CoA in eight
enzymatic steps, shown in FIG. 20. In this route, glutamate is acylated
to glutamyl-CoA by a CoA transferase or ligase (Step A of FIG. 20).
Glutamyl-CoA and acetyl-CoA are joined by a beta-ketothiolase to form the
C7 compound 3-oxo-6-aminopimeloyl-CoA (Step B of FIG. 20). The 3-oxo
group of this product is then reduced and dehydrated, resulting in
6-amino-7-carboxyhept-2-enoyl-CoA (Steps C and D of FIG. 20). An
enoyl-CoA reductase reduces the double bond, forming 6-aminopimeloyl-CoA
(Step E of FIG. 20). 6-Aminopimeloyl-CoA is then converted to
2-amino-7-oxoheptanoate by a CoA-dependent aldehyde dehydrogenase (Step
F). Transamination of the aldehyde to an amine yields homolysine (Step G
of FIG. 20). Finally, HMDA is formed as the decarboxylation product of
homolysine (Step H of FIG. 20). The maximum theoretical HMDA yield for
this pathway is 0.67 moles of HMDA per mole of glucose utilized. Yield
calculations assume aerobic conditions and the utilization of a CoA
transferase in Step A.

[0584] HMDA can also be produced from glutaryl-CoA by several routes.
Exemplary routes for HMDA production are shown in FIG. 21. Glutaryl-CoA
is a common metabolic intermediate in organisms that metabolize aromatic
compounds. In the disclosed pathways to HMDA, glutaryl-CoA is first
condensed with acetyl-CoA by a beta-ketothiolase to form
3-oxopimeloyl-CoA (Step A of FIG. 21). The CoA moiety of
3-oxopimeloyl-CoA is removed by a CoA hydrolase, transferase and ligase
(Step B of FIG. 21). Several alternate routes for converting
3-oxopimelate to HMDA are outlined in FIG. 21 and described herein. The
final step of all routes to HMDA entails decarboxylation of homolysine
(Step S of FIG. 21).

[0585] One route entails conversion of 3-oxopimelate to
3-oxo-1-carboxyheptanal. This conversion can be catalyzed by an ATP- and
NAD(P)H dependent enzyme with 3-oxopimelate reductase activity (Step C of
FIG. 21), or alternately can proceed through activated intermediates
5-oxopimeloyl-CoA (Steps H, I of FIG. 21) or 5-oxopimeloyl-phosphonate
(Steps F, G of FIG. 21). Once formed, 3-oxo-1-carboxyheptanal is
transaminated at the 3-position (Step AB of FIG. 21) or 7-position (Step
D of FIG. 21). Subsequent transamination of 3-oxo-7-aminoheptanoate (Step
E of FIG. 21) or 3-amino-7-oxoheptanoate (Step Z of FIG. 21) yields
3,7-diaminoheptanoate. An enzyme with 3,7-diaminoheptanoate
2,3-aminomutase activity then forms homolysine (Step R of FIG. 21), which
is decarboxylated to HMDA (Step S of FIG. 21).

[0586] In an alternate route, 3-oxopimelate is transaminated to
3-aminopimelate (Step J of FIG. 21). 3-Aminopimelate is then converted to
3-amino-7-oxoheptanoate directly (Step O of FIG. 21) or via a CoA (Steps
K, L of FIG. 21) or phosphonic acid (Steps M, N of FIG. 21) intermediate.
3-Amino-7-oxoheptanoate is subsequently converted to
2-amino-7-oxoheptanoate by a 2,3-aminomutase (Step P of FIG. 21).
2-Amino-7-oxoheptanoate is converted to homolysine by an aminotransferase
or aminating oxidoreductase. Alternately, 3-amino-7-oxoheptanoate is
first transaminated (Step Z of FIG. 21) and then converted to homolysine
by an aminomutase (Step R of FIG. 21).

[0587] 3-Aminopimelate can be converted to 2-aminopimelate by a
2,3-aminomutase enzyme (Step T of FIG. 21). An HMDA pathway involving
this intermediate requires reduction of the 7-carboxylic acid to an
aldehyde. This reduction is catalyzed by a bifunctional reductase (Step W
of FIG. 21) or by two enzymes that proceed through a CoA (Steps V, Y of
FIG. 21) or phosphonic acid (Steps U, X of FIG. 21) intermediate. The
product, 2-amino-7-oxoheptanoate is converted to HMDA as described above.

[0588] Two routes for producing HMDA from pyruvate and 4-aminobutanal are
shown in FIG. 22. The routes achieve a maximum yield of 0.67 moles of
HMDA per mole glucose utilized (0.43 g/g) under anaerobic and aerobic
conditions. 4-Aminobutanal is naturally derived from ornithine by
decarboxylation to putrescine and subsequent transamination.
4-Aminobutanal can also originate from 4-aminobutanoate. In one pathway,
4-aminobutanal and pyruvate are joined by aldol condensation to form
2-oxo-4-hydroxy-7-aminoheptanoate (Step A of FIG. 22). The condensation
product is subsequently dehydrated (Step B of FIG. 22) and reduced (Step
C of FIG. 22). Transamination of 2-oxo-7-aminoheptanoate yields
homolysine (Step D of FIG. 22). HMDA is the decarboxylation product of
homolysine decarboxylase (Step E of FIG. 22). Alternately, pathway
intermediate 2-oxo-7-aminoheptanoate is decarboxylated to form
6-aminohexanal (Step F of FIG. 22). 6-Aminohexanal is subsequently
converted to HMDA by an aminotransferase or aminating oxidoreductase
(Step G of FIG. 22).

[0589] Several routes for producing HMDA from 2-amino-7-oxosubarate are
shown in FIG. 26. 2-Amino-7-oxosubarate is not known to be a naturally
occurring metabolite. An exemplary route for synthesizing
2-amino-7-oxosubarate is shown in FIG. 27. The pathway originates with
glutamate-5-semialdehyde, a metabolite naturally formed during ornithine
biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three
enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed
with pyruvate by an aldolase (FIG. 27, Step A). The product,
2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and the
resulting alkene is reduced to form 2-amino-7-oxosubarate (FIG. 27, Steps
B/C). In one proposed pathway to HMDA from 2-amino-7-oxosubarate, the
2-oxo acid is first decarboxylated to form 2-amino-7-oxoheptanoate (Step
A of FIG. 26). This product is again decarboxylated, forming
6-aminohexanal (Step B of FIG. 26). Finally, 6-aminohexanal is converted
to HMDA by an aminotransferase or aminating oxidoreductase (Step C of
FIG. 26).

[0590] Alternately, the intermediate 2-amino-7-oxoheptanoate is first
converted to homolysine by an aminotransferase or aminating
oxidoreductase (Step M of FIG. 26). Homolysine is decarboxylated to HMDA
as described previously (Step H of FIG. 26).

[0591] In yet another route, the 2-amino acid group of
2-amino-7-oxosubarate is decarboxylated, yielding 2-oxo-7-aminoheptanoate
(Step I of FIG. 26). This product can then be further decarboxylated to
6-aminohexanal (Step G of FIG. 26) or transaminated to homolysine (Step J
of FIG. 26). Homolysine or 6-aminohexanal is then converted to HMDA as
described previously.

[0592] In yet another route, the 2-oxo group of 2-amino-7-oxosubarate is
converted to an amino group, forming 2,7-diaminosubarate (Step K of FIG.
26). Two subsequent decarboxylations yield HMDA (Steps L, H of FIG. 26).

[0593] Described herein is the generation of a microbial organism that has
been engineered to produce HMDA from pyruvate and 4-aminobutanal, as
shown in FIG. 22 (Steps A-E). This example also teaches a method for
engineering a strain that overproduces HMDA.

[0594] Escherichia coli is used as a target organism to engineer a
HMDA-producing pathway as shown in FIG. 22. E. coli provides a good host
for generating a non-naturally occurring microorganism capable of
producing HMDA. E. coli is amenable to genetic manipulation and is known
to be capable of producing various products, like ethanol, acetic acid,
formic acid, lactic acid, and succinic acid, effectively under anaerobic,
microaerobic or aerobic conditions.

[0595] An E. coli strain is engineered to produce HMDA from 4-aminobutanal
via the route outlined in FIG. 22. For the first stage of pathway
construction, genes encoding enzymes to transform 4-aminobutanal and
pyruvate to homolysine (FIG. 3, Steps A-D) are assembled onto vectors. In
particular, the genes hpcH (CAA87759), hpcG (CAA57202), enr
(YP--430895) and lysN ( ) genes encoding
2-oxo-4-hydroxy-7-aminoheptanoate aldolase,
2-oxo-4-hydroxy-7-aminoheptanoate dehydratase, 2-oxo-7-aminohept-3-enoate
reductase and 2-oxo-7-aminoheptanoate aminotransferase, respectively, are
cloned into the pZE13 vector (Expressys, Ruelzheim, Germany), under the
control of the PA1/lacO promoter. The plasmid is transformed into E. coli
strain MG1655 to express the proteins and enzymes required for HMDA
synthesis from 4-aminobutanal. E. coli naturally encodes two lysine
decarboxylase enzymes which convert homolysine to HMDA.

[0596] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the art
(see, for example, Sambrook et al., supra, 2001). The expression of HMDA
pathway genes is corroborated using methods well known in the art for
determining polypeptide expression or enzymatic activity, including for
example, Northern blots, PCR amplification of mRNA and immunoblotting.
Enzymatic activities of the expressed enzymes are confirmed using assays
specific for the individually activities. The ability of the engineered
E. coli strain to produce HMDA through this pathway is confirmed using
HPLC, gas chromatography-mass spectrometry (GCMS) or liquid
chromatography-mass spectrometry (LCMS).

[0597] Microbial strains engineered to have a functional HMDA synthesis
pathway from 4-aminobutanal are further augmented by optimization for
efficient utilization of the pathway. Briefly, the engineered strain is
assessed to determine whether any of the exogenous genes are expressed at
a rate limiting level. Expression is increased for any enzymes expressed
at low levels that can limit the flux through the pathway by, for
example, introduction of additional gene copy numbers.

[0598] After successful demonstration of enhanced HMDA production via the
activities of the exogenous enzymes, the genes encoding these enzymes are
inserted into the chromosome of a wild type E. coli host using methods
known in the art. Such methods include, for example, sequential single
crossover (Gay et al., J. Bacteriol. 3:153 (1983)). and Red/ET methods
from GeneBridges (Zhang et al., European Patent Application No. 01117
(2001))). Chromosomal insertion provides several advantages over a
plasmid-based system, including greater stability and the ability to
co-localize expression of pathway genes.

[0599] To generate better producers, metabolic modeling is utilized to
optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway (see, for
example, U.S. patent publications US 2002/0012939, US 2003/0224363, US
2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US
2004/0009466, and in U.S. Pat. No. 7,127,379). Modeling analysis allows
reliable predictions of the effects on cell growth of shifting the
metabolism towards more efficient production of HMDA. One modeling method
is the bilevel optimization approach, OptKnock (Burgard et al.,
Biotechnol. Bioengineer. 84:647-657 (2003)), which is applied to select
gene knockouts that collectively result in better production of HMDA.
Adaptive evolution also can be used to generate better producers of, for
example, the 2-oxo-4-hydroxy-7-aminoheptanoate intermediate or the HMDA
product. Adaptive evolution is performed to improve both growth and
production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058
(2004); Alper et al., Science 314:1565-1568 (2006)). Based on the
results, subsequent rounds of modeling, genetic engineering and adaptive
evolution can be applied to the HMDA producer to further increase
production.

[0600] For large-scale production of HMDA, the above HMDA
pathway-containing organism is cultured in a fermenter using a medium
known in the art to support growth of the organism under anaerobic
conditions. Fermentations are performed in either a batch, fed-batch or
continuous manner. Anaerobic conditions are maintained by first sparging
the medium with nitrogen and then sealing culture vessel (e.g., flasks
can be sealed with a septum and crimp-cap). Microaerobic conditions also
can be utilized by providing a small hole for limited aeration. The pH of
the medium is maintained at a pH of 7 by addition of an acid, such as
H2SO4. The growth rate is determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by monitoring
carbon source depletion over time. Byproducts such as undesirable
alcohols, organic acids, and residual glucose can be quantified by HPLC
(Shimadzu) with an HPX-087 column (BioRad), using a refractive index
detector for glucose and alcohols, and a UV detector for organic acids,
Lin et al., Biotechnol. Bioeng., 775-779 (2005).

Example XXV

Pathways for Production of 6-Aminocaproate from Glutamate, Glutaryl-CoA,
Homolysine, or 2-Amino-7-oxosubarate

[0601] Novel pathways for producing 6-aminocaproate (6-ACA) and related
products are described herein. The candidate enzymes, and associated
risks of implementation are discussed in Example XXVI below.

[0602] This invention is directed, in part, to non-naturally occurring
microorganisms that express genes encoding enzymes that catalyze 6-ACA
production. Successfully engineering these pathways entails identifying
an appropriate set of enzymes with sufficient activity and specificity,
cloning their corresponding genes into a production host, optimizing the
expression of these genes in the production host, optimizing fermentation
conditions, and assaying for product formation following fermentation.

[0603] 6-ACA can be produced from glutamate as a starting molecule.
Glutamate is transformed to 6-aminopimeloyl-CoA as described previously
(FIG. 20, Steps A-E). Removal of the CoA moiety of 6-Aminopimeloyl-CoA by
a CoA hydrolase, transferase or ligase yields 2-aminopimelate (Step I of
FIG. 20). Decarboxylation of this product yields 6-ACA (Step J of FIG.
20).

[0604] 6-ACA can also be produced from glutaryl-CoA as a starting
molecule. In the disclosed pathway to 6-ACA, similar to the HMDA pathway
described above, glutaryl-CoA is first condensed with acetyl-CoA by a
beta-ketothiolase to form 3-oxopimeloyl-CoA (Step A of FIG. 21). The CoA
moiety of 3-oxopimeloyl-CoA is removed by a CoA hydrolase, transferase
and ligase (Step B of FIG. 21). Then 3-oxopimelate is transaminated to
3-aminopimelate (Step J of FIG. 21). 3-Aminopimelate can be converted to
2-aminopimelate by a 2,3-aminomutase enzyme (Step T of FIG. 21)
Aminopimelate can then be decarboxylated to form 6-aminocaproic acid
(Step AA of FIG. 21).

[0605] Homolysine is also an attractive precursor to 6-aminocaproate
(6-ACA) production. Although homolysine is a potentially valuable
precursor, it is not a known metabolic intermediate of any organism.
Under aerobic conditions, oxidation of homolysine by a lysine
2-monooxygenase yields 6-aminohexanamide, which is readily hydrolyzed to
6-ACA in dilute acid or basic solution (FIG. 23).

[0606] 6-ACA can also be produced from 2-amino-7-oxosubarate as a starting
molecule (FIG. 26). 2-Amino-7-oxosubarate is not known to be a naturally
occurring metabolite. An exemplary route for synthesizing
2-amino-7-oxosubarate is shown in FIG. 27. The pathway originates with
glutamate-5-semialdehyde, a metabolite naturally formed during ornithine
biosynthesis. 2-Amino-7-oxosubarate is then synthesized in three
enzymatic steps. In the first step, glutamate-5-semialdehyde is condensed
with pyruvate by an aldolase (FIG. 27, Step A). The product,
2-amino-5-hydroxy-7-oxosubarate is subsequently dehydrated and the
resulting alkene is reduced to form 2-amino-7-oxosubarate (FIG. 27, Steps
B/C). In one proposed route, 2-amino-7-oxosubarate is decarboxylated to
form 2-amino-7-oxoheptanoate (Step A of FIG. 26). The aldehyde of
2-amino-7-oxoheptanoate is oxidized by an oxidoreductase to form
2-aminopimelate (Step D of FIG. 26). 6-ACA is the decarboxylation product
of 2-aminopimelate (Step E of FIG. 26). Alternately, the
2-amino-7-oxoheptanoate intermediate is decarboxylated to form
6-aminohexanal (Step B of FIG. 26), which is transaminated to 6-ACA (Step
F of FIG. 26). In a third proposed route, the 2-amino acid group of
2-amino-7-oxosubarate is decarboxylated, yielding 2-oxo-7-aminoheptanoate
(Step I of FIG. 26). This product can then be further decarboxylated to
6-aminohexanal (Step G of FIG. 26). Finally, 6-aminohexanal is
transaminated to 6-ACA (Step F of FIG. 26).

Example XXVI

Enzyme Classification System for Production of Hexamethylenediamine and
6-Aminocaproic Acid

[0607] This example describes the enzyme classification system for the
exemplary pathways described in Examples XXIV and XXV for production of
hexamethylenediamine or 6-aminocaproate.

[0608] All transformations depicted in FIGS. 20-23 and 26 fall into the
general categories of transformations shown in Table 11. Below is
described a number of biochemically characterized genes in each category.
Specifically listed are genes that can be applied to catalyze the
appropriate transformations in FIGS. 20-23 and 26 when properly cloned
and expressed.

[0609] Table 11 shows the enzyme types useful to convert common central
metabolic intermediates into 6-aminocaproate and hexamethylenediamine.
The first three digits of each label correspond to the first three Enzyme
Commission number digits which denote the general type of transformation
independent of substrate specificity.

[0620] An additional enzyme type that converts an acyl-CoA to its
corresponding aldehyde is malonyl-CoA reductase which transforms
malonyl-CoA to malonic semialdehyde. Malonyl-CoA reductase is a key
enzyme in autotrophic carbon fixation via the 3-hydroxypropionate cycle
in thermoacidophilic archael bacteria (Berg et al., Science.
318:1782-1786 (2007); and Thauer et al., Science. 318:1732-1733 (2007)).
The enzyme utilizes NADPH as a cofactor and has been characterized in
Metallosphaera and Sulfolobus spp (Alber et al., J. Bacteriol.
188:8551-8559 (2006); and Hugler et al., J. Bacteriol. 184:2404-2410
(2002)). The enzyme is encoded by Msed--0709 in Metallosphaera
sedula (Alber et al., J. Bacteriol. 188:8551-8559 (2006); and Berg et
al., Science. 318:1782-1786 (2007)). A gene encoding a malonyl-CoA
reductase from Sulfolobus tokodaii was cloned and heterologously
expressed in E. coli (Alber et al., J. Bacteriol. 188:8551-8559 (2006)).
This enzyme has also been shown to catalyze the conversion of
methylmalonyl-CoA to its corresponding aldehyde (WO/2007/141208).
Although the aldehyde dehydrogenase functionality of these enzymes is
similar to the bifunctional dehydrogenase from Chloroflexus aurantiacus,
there is little sequence similarity. Both malonyl-CoA reductase enzyme
candidates have high sequence similarity to aspartate-semialdehyde
dehydrogenase, an enzyme catalyzing the reduction and concurrent
dephosphorylation of aspartyl-4-phosphate to aspartate semialdehyde.
Additional gene candidates can be found by sequence homology to proteins
in other organisms including Sulfolobus solfataricus and Sulfolobus
acidocaldarius. Yet another candidate for CoA-acylating aldehyde
dehydrogenase is the ald gene from Clostridium beijerinckii (Toth et al.,
Appl Environ. Microbiol. 65:4973-4980 (1999)). This enzyme has been
reported to reduce acetyl-CoA and butyryl-CoA to their corresponding
aldehydes. This gene is very similar to cutE that encodes acetaldehyde
dehydrogenase of Salmonella typhimurium and E. coli (Toth et al., Appl
Environ. Microbiol. 65:4973-4980 (1999)).

[0625] Several transformations in FIG. 21 require the conversion of an
acid to an aldehyde (FIG. 21, Steps C, O, W). Such a transformation is
thermodynamically unfavorable and typically requires energy-rich
cofactors and multiple enzymatic steps. For example, in butanol
biosynthesis conversion of butyrate to butyraldehyde is catalyzed by
activation of butyrate to its corresponding acyl-CoA by a CoA transferase
or ligase, followed by reduction to butyraldehyde by a CoA-dependent
aldehyde dehydrogenase. Alternately, an acid can be activated to an
acyl-phosphate and subsequently reduced by a phosphate reductase. Direct
conversion of the acid to aldehyde by a single enzyme is catalyzed by an
enzyme in the 1.2.1 family. Exemplary enzymes that catalyze these
transformations include carboxylic acid reductase, alpha-aminoadipate
reductase and retinoic acid reductase.

[0627] An enzyme with similar characteristics, alpha-aminoadipate
reductase (AAR, EC 1.2.1.31), participates in lysine biosynthesis
pathways in some fungal species. This enzyme naturally reduces
alpha-aminoadipate to alpha-aminoadipate semialdehyde. The carboxyl group
is first activated through the ATP-dependent formation of an adenylate
that is then reduced by NAD(P)H to yield the aldehyde and AMP. Like CAR,
this enzyme utilizes magnesium and requires activation by a PPTase.
Enzyme candidates for AAR and its corresponding PPTase are found in
Saccharomyces cerevisiae (Morris et al., Gene 98:141-145 (1991)), Candida
albicans (Guo et al., Mol. Genet. Genomics 269:271-279 (2003)), and
Schizosaccharomyces pombe (Ford et al., Curr. Genet. 28:131-137 (1995)).
The AAR from S. pombe exhibited significant activity when expressed in E.
coli (Guo et al., Yeast 21:1279-1288 (2004)). The AAR from Penicillium
chrysogenum accepts S-carboxymethyl-L-cysteine as an alternate substrate,
but did not react with adipate, L-glutamate or diaminopimelate
(Hijarrubia et al., J. Biol. Chem. 278:8250-8256 (2003)). The gene
encoding the P. chrysogenum PPTase has not been identified to date and no
high-confidence hits were identified by sequence comparison homology
searching. Directed evolution or other enzyme engineering methods may be
required to enhance reactivity with the substrates in FIG. 21.

[0629] Three transformations fall into the category of oxidoreductases
that reduce an alkene to an alkane (EC 1.3.1.-). The conversion of
6-amino-7-carboxy-hept-2-enoyl-CoA to 6-aminopimeloyl-CoA (FIG. 20, Step
E),2-oxo-7-aminohept-3-onoate to 2-oxo-7-aminoheptanoate (FIG. 22, Step
C) and 2-amino-5-ene-7-oxosubarate to 2-amino-7-oxosubarate (FIG. 27,
Step C) are catalyzed by a 2-enoate reductase. 2-Enoate reductase enzymes
are known to catalyze the NAD(P)H-dependent reduction of a wide variety
of α, β-unsaturated carboxylic acids and aldehydes (Rohdich,
et al., J. Biol. Chem. 276:5779-5787 (2001)). In the recently published
genome sequence of C. kluyveri, 9 coding sequences for enoate reductases
were reported, out of which one has been characterized (Seedorf et al.,
Proc. Natl. Acad. Sci. U.S. A 105:2128-2133 (2008)). The enr genes from
both C. tyrobutyricum and M. thermoaceticum have been cloned and
sequenced and show 59% identity to each other. The former gene is also
found to have approximately 75% similarity to the characterized gene in
C. kluyveri (Giesel et al., Arch. Microbiol. 135:51-57 (1983)). It has
been reported based on these sequence results that enr is very similar to
the dienoyl CoA reductase in E. coli (fades) (Rohdich, et al., J. Biol.
Chem. 276:5779-5787 (2001)). The Moorella thermoacetica (formerly C.
thermoaceticum) enr gene has also been expressed in a catalytically
active form in E. coli (Ohdich, et al., J Biol. Chem. 276:5779-5787
(2001)).

[0631] Enoyl-CoA reductase enzymes are suitable enzymes for catalyzing the
reduction of 6-amino-7-carboxyhept-2-enoyl-CoA to 6-aminopimeloyl-CoA
(FIG. 20, Step E). One exemplary enoyl-CoA reductase is the gene product
of bcd from C. acetobutylicum (Atsumi et al., Metab Eng. 10:305-311
(2008)); and Boynton et al., J Bacteriol. 178:3015-3024 (1996)), which
naturally catalyzes the reduction of crotonyl-CoA to butyryl-CoA.
Activity of this enzyme can be enhanced by expressing bcd in conjunction
with expression of the C. acetobutylicum etfAB genes, which encode an
electron transfer flavoprotein. An additional candidate for the enoyl-CoA
reductase step is the mitochondrial enoyl-CoA reductase from E. gracilis
(Hoffmeister, et al., J. Biol. Chem. 280:4329-4338 (2005)). A construct
derived from this sequence following the removal of its mitochondrial
targeting leader sequence was cloned in E. coli resulting in an active
enzyme (Hoffmeister, et al., J. Biol. Chem. 280:4329-4338 (2005)). This
approach is well known to those skilled in the art of expressing
eukaryotic genes, particularly those with leader sequences that may
target the gene product to a specific intracellular compartment, in
prokaryotic organisms. A close homolog of this gene, TDE0597 from the
prokaryote Treponema denticola, represents a third enoyl-CoA reductase
which has been cloned and expressed in E. coli (Tucci et al., Febs
Letters 581:1561-1566 (2007)).

[0633] An additional candidate is 2-methyl-branched chain enoyl-CoA
reductase (EC 1.3.1.52), an enzyme catalyzing the reduction of sterically
hindered trans-enoyl-CoA substrates. This enzyme participates in
branched-chain fatty acid synthesis in the nematode Ascarius suum and is
capable of reducing a variety of linear and branched chain substrates
including 2-methylbutanoyl-CoA, 2-methylpentanoyl-CoA, octanoyl-CoA and
pentanoyl-CoA (Duran et al., J Biol. Chem. 268:22391-22396 (1993))). Two
isoforms of the enzyme, encoded by genes acad1 and acad, have been
characterized.

[0635] Several reactions in FIGS. 20-23 require the conversion of ketones
or aldehydes to amine groups. Such a transformation can be accomplished
by aminating oxidoreductases in the EC class 1.4.1. Enzymes in this EC
class catalyze the oxidative deamination of amino groups with NAD+ or
NADP+ as acceptor, and the reactions are typically reversible.

[0640] In Step A of FIG. 21, Glutaryl-CoA and acetyl-CoA are condensed to
form 3-oxopimeloyl-CoA by oxopimeloyl-CoA:glutaryl-CoA acyltransferase, a
beta-ketothiolase (EC 2.3.1.16). An enzyme catalyzing this transformation
is found in Ralstonia eutropha (formerly known as Alcaligenes eutrophus),
encoded by genes bktB and bktC (Haywood et al., FEMS Microbiology Letters
52:91-96 (1988); and Slater et al., J. Bacteriol. 180:1979-1987 (1998)).
The sequence of the BktB protein is known; however, the sequence of the
BktC protein has not been reported. The pim operon of Rhodopseudomonas
palustris also encodes a beta-ketothiolase, encoded by pimB, predicted to
catalyze this transformation in the degradative direction during
benzoyl-CoA degradation (Harrison et al., Microbiology 151:727-736
(2005)). A beta-ketothiolase enzyme candidate in S. aciditrophicus was
identified by sequence homology to bktB (43% identity, evalue=1e-93).

[0641] Beta-ketothiolase enzymes catalyzing the formation of
beta-ketovalerate from acetyl-CoA and propionyl-CoA may also be able to
catalyze the formation of 3-oxopimeloyl-CoA. Zoogloea ramigera possesses
two ketothiolases that can form β-ketovaleryl-CoA from propionyl-CoA
and acetyl-CoA and R. eutropha has a β-oxidation ketothiolase that
is also capable of catalyzing this transformation (Gruys et al., U.S.
Pat. No. 5,958,745 (1999)). The sequences of these genes or their
translated proteins have not been reported, but several candidates in R.
eutropha, Z. ramigera, or other organisms can be identified based on
sequence homology to bktB from R. eutropha. These include:

[0644] A beta-ketothiolase is also required to condense glutamyl-CoA and
acetyl-CoA (FIG. 20, Step B). This transformation is not known to occur
naturally. The beta-ketothiolase candidates described above are also
exemplary candidates for catalyzing this transformation.

[0645] 2.6.1.a Aminotransferase

[0646] Several reactions in FIGS. 20-26 are catalyzed by aminotransferases
in the EC class 2.6.1. Such enzymes reversibly transfer amino groups from
aminated donors to acceptors such as pyruvate and alpha-ketoglutarate.

[0651] Several aminotransferases transaminate the amino groups of 2-oxo
acids to form amino acids. Such an enzyme is required for the
transamination of 2-oxo-7-aminoheptanoate to homolysine (FIG. 22, Step D;
FIG. 26, Step M) and 2-amino-7-oxosubarate to 2,7-diaminosubarate (FIG.
26, Step K). A promising enzyme candidate is alpha-aminoadipate
aminotransferase (EC 2.6.1.39), an enzyme that participates in lysine
biosynthesis and degradation in some organisms. This enzyme interconverts
2-aminoadipate and 2-oxoadipate, using alpha-ketoglutarate as the amino
acceptor. Gene candidates are found in Homo sapiens (Okuno et al., Enzyme
Protein 47:136-148 (1993)) and Thermus thermophilus (Miyazaki et al.,
Microbiology 150:2327-2334 (2004)). The Thermus thermophilus enzyme,
encoded by lysN, is active with several alternate substrates including
oxaloacetate, 2-oxoisocaproate, 2-oxoisovalerate, and
2-oxo-3-methylvalerate.

[0661] The hydrolysis of 6-aminopimeloyl-CoA to 6-aminopimelate (FIG. 20,
Step I) is carried out by an acyl CoA hydrolase enzyme in the 3.1.2
family. An enzyme catalyzing this transformation has not been
demonstrated to date. Several eukaryotic acetyl-CoA hydrolases (EC
3.1.2.1) have broad substrate specificity and thus represent suitable
candidate enzymes for hydrolyzing 6-aminopimelate. For example, the
enzyme from Rattus norvegicus brain (Robinson et al., Res. Commun.
71:959-965 (1976)) can react with butyryl-CoA, hexanoyl-CoA and
malonyl-CoA. Though its sequence has not been reported, the enzyme from
the mitochondrion of the pea leaf also has a broad substrate specificity,
with demonstrated activity on acetyl-CoA, propionyl-CoA, butyryl-CoA,
palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA (Zeiher et al.,
Plant. Physiol. 94:20-27 (1990)). The acetyl-CoA hydrolase, ACH1, from S.
cerevisiae represents another candidate hydrolase (Buu et al., J. Biol.
Chem. 278:17203-17209 (2003)).

[0663] Yet another candidate hydrolase is the glutaconate CoA-transferase
from Acidaminococcus fermentans. This enzyme was transformed by
site-directed mutagenesis into an acyl-CoA hydrolase with activity on
glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA (Mack et al., FEBS. Lett.
405:209-212 (1997)). This suggests that the enzymes encoding
succinyl-CoA:3-ketoacid-CoA transferases and acetoacetyl-CoA:acetyl-CoA
transferases may also serve as candidates for this reaction step but
would require certain mutations to change their function.

[0668] Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broad
substrate range and has been the target of enzyme engineering studies.
The enzyme from Pseudomonas putida has been extensively studied and
crystal structures of this enzyme are available (Hasson et al.,
Biochemistry 37:9918-9930 (1998); and Polovnikova et al., Biochemistry
42:1820-1830 (2003)). Site-directed mutagenesis of two residues in the
active site of the Pseudomonas putida enzyme altered the affinity (Km) of
naturally and non-naturally occurring substrates (Siegert et al., Protein
Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been
further modified by directed engineering (Lingen et al., Protein Eng
15:585-593 (2002); and Lingen et al., Chembiochem. 4:721-726 (2003)). The
enzyme from Pseudomonas aeruginosa, encoded by mdlC, has also been
characterized experimentally (Barrowman et al., FEMS Microbiology Letters
34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri,
Pseudomonas fluorescens and other organisms can be inferred by sequence
homology or identified using a growth selection system developed in
Pseudomonas putida (Henning et al., Appl. Environ. Microbiol.
72:7510-7517 (2006)).

[0669] A third enzyme capable of decarboxylating 2-oxoacids is
alpha-ketoglutarate decarboxylase (KGD). The substrate range of this
class of enzymes has not been studied to date. The KDC from Mycobacterium
tuberculosis (Tian et al., Proc Natl Acad Sci U S. A 102:10670-10675
(2005)) has been cloned and functionally expressed in other internal
projects at Genomatica. However, it is not an ideal candidate for strain
engineering because it is large (-130 kD) and GC-rich. KDC enzyme
activity has been detected in several species of rhizobia including
Bradyrhizobium japonicum and Mesorhizobium loti (Green et al., J.
Bacteriol. 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have
not been isolated in these organisms, the genome sequences are available
and several genes in each genome are annotated as putative KDCs. A KDC
from Euglena gracilis has also been characterized but the gene associated
with this activity has not been identified to date (Shigeoka and Nakano,
Arch. Biochem. Biophys. 288:22-28 (1991)). The first twenty amino acids
starting from the N-terminus were sequenced MTYKAPVKDVKFLLDKVFKV (SEQ ID
NO: 1) (Shigeoka and Nakano, Arch. Biochem. Biophys. 288:22-28 (1991)).
The gene could be identified by testing candidate genes containing this
N-terminal sequence for KDC activity.

[0670] A fourth candidate enzyme for catalyzing this reaction is branched
chain alpha-ketoacid decarboxylase (BCKA). This class of enzyme has been
shown to act on a variety of compounds varying in chain length from 3 to
6 carbons (Oku and Kaneda, J Biol. Chem. 263:18386-18396 (1988); and Smit
et al., Appl Environ Microbiol. 71:303-311 (2005)). The enzyme in
Lactococcus lactis has been characterized on a variety of branched and
linear substrates including 2-oxobutanoate, 2-oxohexanoate,
2-oxopentanoate, 3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and
isocaproate isocaproate (Smit et al., Appl Environ Microbiol. 71:303-311
(2005)). The enzyme has been structurally characterized (Berg et al.,
Science. 318:1782-1786 (2007)). Sequence alignments between the
Lactococcus lactis enzyme and the pyruvate decarboxylase of Zymomonas
mobilus indicate that the catalytic and substrate recognition residues
are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357
(2005)), so this enzyme would be a promising candidate for directed
engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was
detected in Bacillus subtilis; however, this activity was low (5%)
relative to activity on other branched-chain substrates (Oku and Kaneda,
J Biol. Chem. 263:18386-18396 (1988)) and the gene encoding this enzyme
has not been identified to date. Additional BCKA gene candidates can be
identified by homology to the Lactococcus lactis protein sequence. Many
of the high-scoring BLASTp hits to this enzyme are annotated as
indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase
(IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate
to indoleacetaldehyde in plants and plant bacteria.

[0673] The condensation of pyruvate with 4-aminobutanal (FIG. 22, Step A)
or glutamate-5-semialdehyde (FIG. 27, Step A) is catalyzed by an aldehyde
lyase in the EC class 4.1.2. A variety of aldehyde lyase enzymes utilize
pyruvate as an acceptor; however, none have been demonstrated to utilize
4-aminobutanal or glutamate-5-semialdehyde as a donor. The enzyme
4-hydroxy-2-oxopimelate (HODH) aldolase (EC 4.1.2.-), condenses succinic
semialdehyde and pyruvate to catalyze the formation of
4-hydroxy-2-oxopimelate. This enzyme is a divalent metal ion-dependent
class II aldolase, catalyzing the final step of 4-hydroxyphenylacetic
acid degradation in E. coli C, E. coli W, and other organisms. In the
native context, the enzyme functions in the degradative direction. The
reverse (condensation) reaction is thermodynamically unfavorable; however
the equilibrium can be shifted through coupling HODH aldolase with
downstream pathway enzymes that work efficiently on reaction products.
Such strategies have been effective for shifting the equilibrium of other
aldolases in the condensation direction (Nagata et al., Appl Microbiol
Biotechnol 44:432-438 (1995); and Pollard et al., Appl Environ.
Microbiol. 64:4093-4094 (1998)). The E. coli C enzyme, encoded by hpcH,
is able to condense a range of aldehyde acceptors with pyruvate and has
recently been crystallized (Rea et al., J Mol. Biol. 373:866-876 (2007);
and

[0682] Another enzyme candidate is citramalate hydrolyase (EC 4.2.1.34),
an enzyme that naturally dehydrates 2-methylmalate to mesaconate. This
enzyme has been studied in Methanocaldococcus jannaschii in the context
of the pyruvate pathway to 2-oxobutanoate, where it has been shown to
have a broad substrate specificity (Drevland et al., J Bacteriol.
189:4391-4400 (2007)). This enzyme activity was also detected in
Clostridium tetanomorphum, Morganella morganii, Citrobacter amalonaticus
where it is thought to participate in glutamate degradation (Kato et al.,
Arch. Microbiol. 168:457-463 1997)). The M. jannaschii protein sequence
does not bear significant homology to genes in these organisms.

[0684] Several reactions in FIG. 21 involve shifting a secondary amine
from the 3- to the 2-position (FIG. 21, Steps P, R, T). A promising
enzyme candidate for catalyzing these transformations is lysine
2,3-aminomutase (EC 5.4.3.2), an enzyme that naturally converts lysine to
(3S)-3,6-diaminohexanoate, reversibly shifting an amine group from the 2-
to the 3-position. The enzyme is found in bacteria that ferment lysine to
acetate and butyrate, including Fusobacterium nucleatum (kamA) (Barker et
al., J. Bacteriol. 152:201-207 (1982)) and Clostridium subterminale
(kamA) (Chirpich et al., J. Biol. Chem. 245:1778-1789 (1970)). The enzyme
from Clostridium subterminale has been crystallized (117). An enzyme
encoding this function is also encoded by yodO in Bacillus subtilis (Chen
et al., Biochem. J. 348 Pt 3:539-549 (2000)). The enzyme utilizes
pyridoxal 5'-phosphate as a cofactor, requires activation by
S-adenosylmethoionine, and is stereoselective for L-lysine. The enzyme
has not been shown to react with alternate substrates, so directed
evolution or other engineering methods may be required for this enzyme to
react with the non-natural substrates 3-amino-7-oxohexanoate,
3,7-diaminoheptanoate and 3-aminopimelate. For example, Cargill has
developed a novel 2,3-aminomutase enzyme derived from
lysine-2,3-aminomutase that converts L-alanine to β-alanine (Liao et
al., United States Patent 20050221466 (2005)).

[0685] Other enzymes with 2,3-aminomutase activity include tyrosine
2,3-aminomutase (EC 5.4.3.6) and leucine 2,3-aminomutase (EC 5.4.3.7).
Tyrosine 2,3-aminomutase participates in tyrosine biosynthesis,
reversibly converting tyrosine to 3-amino-3-(4-hydroxyphenyl)-propionoate
by shifting an amine from the 2- to the 3-position. In Streptomyces
globisporus the enzyme has also been shown to react with tyrosine
derivatives (Christenson et al., Biochemistry 42:12708-12718 (2003));
however, the sequence of this enzyme is not yet available. Leucine
2,3-aminomutase converts L-leucine to beta-leucine during leucine
biosynthesis and degradation. A leucine 2,3-aminomutase-specific assay
detected enzyme activity in many organisms (Poston et al., Methods
Enzymol. 166:130-135 (1988)) but genes encoding the enzyme have not been
identified to date.

[0686] 6.2.1.a Acid-Thiol Ligase

[0687] The activation of carboxylic acids to acyl-CoA derivatives is
catalyzed by CoA acid-thiol ligases or CoA synthetases in the EC class
6.2.1 (the terms ligase, synthetase, and synthase are used herein
interchangeably and refer to the same enzyme class). Such enzymes couple
the energetic cost of thioester bond formation to the hydrolysis of ATP
into ADP or AMP. Several ADP-forming CoA ligases have been demonstrated
to react in the reverse direction, removing the CoA moiety from acyl-CoA
molecules and concomitantly forming ATP. Reversible CoA ligases are
required to de-acylate 6-aminopimeloyl-CoA (FIG. 20, Step I) and
3-oxopimeloyl-CoA (FIG. 21, Step B), whereas AMP or ADP forming ligases
can acylate 3-oxopimelate (FIG. 21, Step H), 3-aminopimelate (FIG. 21,
Step K) and 2-aminopimelate (FIG. 21, Step V). Enzymes catalyzing these
exact transformations have not been characterized to date; however,
several enzymes with broad substrate specificities have been described in
the literature.

[0688] ADP-forming acetyl-CoA synthetase (ACD, EC 6.2.1.13) is an enzyme
that couples the conversion of acyl-CoA esters to their corresponding
acids with the concomitant synthesis of ATP. ACD I from Archaeoglobus
fulgidus, encoded by AF1211, was shown to operate on a variety of linear
and branched-chain substrates including isobutyrate, isopentanoate, and
fumarate (Musfeldt et al., J. Bacteriol. 184:636-644 (2002)). A second
reversible ACD in Archaeoglobus fulgidus, encoded by AF1983, was also
shown to have a broad substrate range with high activity on cyclic
compounds phenylacetate and indoleacetate (Musfeldt et al., J. Bacteriol.
184:636-644 (2002)). The enzyme from Haloarcula marismortui (annotated as
a succinyl-CoA synthetase) accepts propionate, butyrate, and
branched-chain acids (isovalerate and isobutyrate) as substrates, and was
shown to operate in the forward and reverse directions (Brasen et al.,
Arch. Microbiol. 182:277-287 (2004)). The ACD encoded by PAE3250 from
hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest
substrate range of all characterized ACDs, reacting with acetyl-CoA,
isobutyryl-CoA (preferred substrate) and phenylacetyl-CoA (Brasen et al.,
Arch. Microbiol. 182:277-287 (2004)). Directed evolution or engineering
can be used to modify this enzyme to operate at the physiological
temperature of the host organism. The enzymes from A. fulgidus, H.
marismortui and P. aerophilum have all been cloned, functionally
expressed, and characterized in E. coli (Brasen et al., Arch. Microbiol.
182:277-287 (2004); and Musfeldt et al., J. Bacteriol. 184:636-644
(2002)). An additional candidate is the enzyme encoded by sucCD in E.
coli, which naturally catalyzes the formation of succinyl-CoA from
succinate with the concomitant consumption of one ATP, a reaction which
is reversible in vivo (Buck et al., Biochemistry 24:6245-6252 (1985)).

Addtitional Pathways for Production of Hexamethylenediamine from
6-Aminocaproate

[0691] FIG. 24 provides additional pathways for HMDA production and is
further to FIG. 13 and Example XX above. Arrows for Steps O and P
indicate the direct conversion of 6-aminocaproate and
6-acetamidohexanoate to 6-aminocaproic semialdehyde and
6-acetamidohexanal, respectively. These reactions are catalyzed by a
reductase in EC class 1.2.1.e. For a description of enzyme candidates,
see Example XXVI (EC 1.2.1.e).

Example XXVIII

Pathways for Production of 6-Aminocaproate from Adipate

[0692] FIG. 25 provides additional pathways for 6-ACA production and is
further to FIG. 10 and Example XVI above. The conversion of adipate to
adipate semialdehyde (FIG. 25, Step X) is catalyzed by an enzyme with
adipate reductase functionality. Adipate kinase catalyzes the formation
of adipylphosphate from adipate (FIG. 25, Step Y). Adipate semialdehyde
is formed from adipylphosphate by adipylphosphate reductase (FIG. 25,
Step Z). Enzyme candidates for catalyzing these transformations are
described in Example XXVI.

Example XXIX

Pathway for Production of Levulinic Acid

[0693] Levulinic acid (LA), also known as 4-oxopentanoic acid and
4-ketovaleric acid, is a precursor to nylon-like polymers, synthetic
rubbers and plastics. It is also a precursor of other commodity chemicals
such as methyltetrahydrofuran, valerolactone and ethyl levulinate. Other
potential applications include use as a fuel extender and a biodegradable
herbicide/pesticide. It is traditionally prepared by treating cellulosic
biomass with strong acids such as hydrochloric and sulfuric acids. This
process has the disadvantages of low LA yield and numerous byproducts.
More recently, the Biofine Process was developed which converts
cellulosic biomass into LA, formic acid and furfural at a yield of 70%
the theoretical maximum (Hayes et al., "The biofine process-production of
levulinic acid, furfural and formic acid from lignocellulosic feedstock"
p. 139-164. In Biorefineries: Industrial Processes and Products. Wiley,
Weinheim, Germany (2006)). Described herein is a process for selectively
producing LA from sugar or syngas feedstocks using a microbial organism.

[0694] The maximum theoretical yield of LA from glucose is 1.45 moles of
LA per mole glucose utilized (0.938 g/g), according to the following
equation:

Glucose(C6H12O2)+1.27CO2→1.45LA(C5H8O3)+0.18H2O

[0695] LA is produced from the central metabolites succinyl-CoA and
acetyl-CoA in three enzymatic steps. In the first step, acetyl-CoA and
succinyl-CoA are condensed by a beta-ketothiolase to form 3-oxoadipyl-CoA
(Step A of FIG. 25). The CoA moiety is subsequently removed by a CoA
hydrolase, transferase or ligase (Steps E/F/G of FIG. 25). In the final
step of the pathway, 3-oxoadipate is decarboxylated to LA (Step AA of
FIG. 25).

[0696] The decarboxylation of 3-oxoadipate to LA can occur enzymatically
or spontaneously. In E. coli, several 3-oxoacids produced during amino
acid biosynthesis have been shown to undergo spontaneous decarboxylation
(Boylan et al., Biochem. Biophys. Res Commun. 85:190-197 (1978)). An
enzyme catalyzing the decarboxylation of 3-oxoadipate to LA has not been
demonstrated to our knowledge. An exemplary enzyme candidate catalyzing a
similar reaction is acetoacetate decarboxylase (EC 4.1.1.4). The
acetoacetate decarboxylase from Clostridium acetobutylicum, encoded by
adc, has a broad substrate specificity and has been shown to
decarboxylate 3-oxopentanoate, 2-oxo-3-phenylpropionic acid and
2-methyl-3-oxobutyrate (Benner et al., J. Am. Chem. Soc. 103:993-994
(1981) and Rozzel et al., J. Am. Chem. Soc. 106:4937-4941 (1984)). An
acetoacetate decarboylase has also been characterized in Clostridium
beijerinckii (Ravagnani et al., Mol. Microbiol. 37:1172-1185 (2000)). The
acetoacetate decarboxylase from Bacillus polymyxa, characterized in
cell-free extracts, also has a broad substrate specificity for 3-keto
acids and can decarboxylate 3-oxopentanoate (Matiasek et al., Curr.
Microbiol. 42:276-281 (2001)). The gene encoding this enzyme has not been
identified to date and the genome sequence of B. polymyxa is not yet
available. Another adc is found in Clostridium saccharoperbutylacetonicum
(Kosaka, et al., Biosci. Biotechnol Biochem. 71:58-68 (2007)).

[0698] Described below in more detail are sets of enzymatic activities
that can be reduced by appropriate gene disruptions or deletions in a
production host engineered to contain the adipate, 6-aminocaproic acid
(6-ACA) and hexamethylenediamine (HMDA) production pathways, for example,
pathways using succinyl CoA and acetyl CoA as precursors.

[0699] OptKnock is a bilevel computational framework formulated with the
overall objective of developing genetically stable overproducing
microorganisms. Specifically, the framework examines the complete network
of a microorganism in order to suggest genetic manipulations that force
the desired biochemical to become an obligatory byproduct of cell growth.
By coupling biochemical production with cell growth through strategically
placed gene disruptions or deletions, the growth selection pressures
imposed on the engineered strains after long periods of time in a
bioreactor lead to improvements in performance as a result of the
compulsory growth-coupled biochemical production. Lastly, in the case of
a gene deletion, there is negligible possibility of the designed strains
reverting to their wild-type states because the genes selected by
OptKnock are completely removed from the genome.

[0700] Growth-coupled biochemical production can be visualized in the
context of the biochemical production envelopes of a typical metabolic
network calculated using an in silico model. These limits are obtained by
fixing the uptake rate(s) of the limiting substrate(s) to their
experimentally measured value(s) and calculating the maximum and minimum
rates of biochemical production at each attainable level of growth.
Although exceptions exist, typically the production of a desired
biochemical is in direct competition with biomass formation for
intracellular resources. Thus, enhanced rates of biochemical production
will generally result in sub-maximal growth rates. The knockouts
suggested by OptKnock are designed to restrict the allowable solution
boundaries forcing a change in metabolic behavior from the wild-type
strain. Although the actual solution boundaries for a given strain will
expand or contract as the substrate uptake rate(s) increase or decrease,
each experimental point should lie within its calculated solution
boundary. Plots such as these allow visualization of how close strains
are to their performance limits or, in other words, how much room is
available for improvement. The OptKnock framework has been used to
identify promising gene deletion strategies for biochemical
overproduction and establishes a systematic framework that will naturally
encompass future improvements in metabolic and regulatory modeling
frameworks.

[0701] Described below are sets of enzyme activities that should be
absent, attenuated, or eliminated for creating host organisms that
achieve growth-coupled adipate, 6-ACA or HMDA production upon the
addition of the biosynthetic pathway that proceeds through succinyl-CoA
and acetyl-CoA. To enumerate all potential strategies, an optimization
technique, termed integer cuts, has been implemented which entails
iteratively solving the OptKnock problem with the incorporation of an
additional constraint referred to as an integer cut at each iteration.

[0702] The OptKnock algorithm was used to identify designs based on a
stoichiometric model of Escherichia coli metabolism. Assumptions include
(i) a glucose uptake rate of 10 mmol/gdw/hr; (ii) anaerobic or
microaerobic conditions; and (iii) a minimum non-growth associated
maintenance requirement of 4 mmol/gdw/hr. Table 12 provides a list of all
the reaction stoichiometries and the associated genes known to be
associated with the reactions identified for deletion in the strategies.
Table 13 provides a list of the metabolite abbreviations, the
corresponding names and locations of all the metabolites that participate
in the reactions listed in Table 12. The growth-coupled productions
designs for adipic acid, 6ACA and HMDA are provided in Tables 14-16. The
product formation rates shown in Tables 14-16 are in mmol/gDCWhr. The
basis glucose uptake rate is 10 mmol/gDCWhr and the biomass formation
rate is shown in units of 1/hr. These tables list the reactions that are
knocked out in a particular strategy, the anticipated product and biomass
yields. Although the designs were identified using a metabolic model of
E. coli metabolism, and the gene names listed are specific to E. coli,
the method of choosing the metabolic engineering strategies and also the
designs themselves are applicable to any HMDA, 6-ACA or adipate-producing
organism. Thus the designs are essentially lists of enzymatic
transformations whose activity is to be either eliminated, attenuated, or
initially absent from a microorganism to provide the growth coupled
production of adipate, 6ACA and HMDA.

[0703] The key criterion for prioritizing the final selection of designs
was the growth-coupled yield of each of the products. To examine this,
production cones were constructed for each strategy by first maximizing
and, subsequently minimizing the product yields at different rates of
biomass formation, as described above. If the rightmost boundary of all
possible phenotypes of the mutant network is a single point, it implies
that there is a unique optimum yield of the product at the maximum
biomass formation rate possible in the network. In other cases, the
rightmost boundary of the feasible phenotypes is a vertical line,
indicating that at the point of maximum biomass the network can make any
amount of the product in the calculated range, including the lowest
amount at the bottommost point of the vertical line. Such designs were
given a lower priority.

[0704] The metabolic engineering strategies described below assume that
the organism can produce adipate, 6-ACA or HMDA via the succinyl CoA and
acetyl-CoA utilizing pathway. The construction of a recombinant host
organism capable of producing these products via the pathway is described
herein.

[0705] Strain Construction:

[0706] In order to validate the computational predictions proposed in this
report, the strains are constructed, evolved, and tested. Escherichia
coli K-12 MG1655 housing the succinyl-CoA-acetyl-CoA pathway serves as
the strain into which the deletions are introduced. The strains are
constructed by incorporating in-frame deletions using homologous
recombination via the λ Red recombinase system of Datsenko and
Wanner (Proc. Natl. Acad. Sci. USA 97(12):6640-6645 2000)). The approach
involves replacing a chromosomal sequence, that is, the gene targeted for
removal, with a selectable antibiotic resistance gene, which itself is
later removed. The knockouts are integrated one by one into the recipient
strain. No antibiotic resistance markers remain after each deletion,
allowing accumulation of multiple mutations in each target strain. The
deletion technology completely removes the gene targeted for removal so
as to substantially reduce the possibility of the constructed mutants
reverting back to the wild-type.

[0707] Shake Flask Characterization:

[0708] As intermediate strains are being constructed, strain performance
is quantified by performing shake flask fermentations. Anaerobic
conditions are obtained by sealing the flasks with a rubber septum and
then sparging the medium with nitrogen. For strains where growth is not
observed under strict anaerobic conditions, microaerobic conditions can
be applied by covering the flask with foil and poking a small hole for
limited aeration. All experiments are performed using M9 minimal medium
supplemented with glucose unless otherwise stated. Pre-cultures are grown
overnight and used as inoculum for a fresh batch culture for which
measurements are taken during exponential growth. The growth rate is
determined by measuring optical density using a spectrophotometer (600
nm), and the glucose uptake rate by monitoring carbon source depletion
over time. The products, ethanol, and organic acids are analyzed by GC-MS
or HPLC using routine procedures. Triplicate cultures are grown for each
strain.

[0709] Batch Fermenter Testing:

[0710] The performance of select strains is tested in anaerobic,
pH-controlled batch fermentations. This allows reliable quantification of
the growth, glucose uptake, and formation rates of all products, as well
as ensure that the accumulation of acidic fermentation products will not
limit cell growth. In addition, it allows accurate determination of
volumetric productivity and yield of product formation, two of the most
important parameters in benchmarking strain performance. Fermentations
are carried out in 1-L bioreactors with 600 mL working volume, equipped
with temperature and pH control. The reactor is continuously sparged with
N2 at approximately 0.5 L/min to ensure that dissolved oxygen (DO)
levels remain below detection levels. The culture medium is the same as
described above, except that the glucose concentration is increased in
accordance with the higher cell density achievable in a fermentation
vessel.

[0711] Chemostat Testing:

[0712] Chemostat experiments are conducted to obtain a direct measure of
how the switch in fermentation mode from batch to continuous affects
product yield and volumetric productivity. The bioreactors described
above using batch mode are operated in chemostat mode through continuous
supply of medium and removal of spent culture. The inlet flow rate is set
to maintain a constant dilution rate of 80% of the maximum growth rate
observed for each strain in batch, and the outlet flow is controlled to
maintain level. Glucose is the limiting nutrient in the medium, and set
to achieve the desired optical density in the vessel.

[0713] Adaptive Evolution:

[0714] The knockout strains are initially expected to exhibit suboptimal
growth rates until their metabolic networks have adjusted to their
missing functionalities. To allow this adjustment, the strains is
adaptively evolved. By subjecting the strains to adaptive evolution,
cellular growth rate becomes the primary selection pressure and the
mutant cells are compelled to reallocate their metabolic fluxes in order
to enhance their rates of growth. This reprogramming of metabolism has
been recently demonstrated for several E. coli mutants that had been
adaptively evolved on various substrates to reach the growth rates
predicted a priori by an in silico model (Fong and Palsson, Nat. Genet.
36(10):1056-1058 (2004). The OptKnock-generated strains are adaptively
evolved in triplicate (running in parallel) due to differences in the
evolutionary patterns witnessed previously in E. coli (Fong and Palsson,
Nat. Genet. 36(10):1056-1058 (2004); Fong et al., J. Bacteriol.
185(21):6400-6408 (2003); Ibarra et al., Nature 420(6912):186-189 (2002))
that could potentially result in one strain having superior production
qualities over the others. Evolutions are run for a period of 2-6 weeks,
depending upon the rate of growth improvement attained. In general,
evolutions are stopped once a stable phenotype is obtained. The
growth-coupled biochemical production concept behind the OptKnock
approach results in the generation of genetically stable overproducers.

[0715] Although described as deletion sets, it is understood, as disclosed
herein, that gene sets can be deleted or disrupted so that encoded gene
product activities are reduced or eliminated. Thus, the gene deletion
sets of Tables 14-16 can be used to delete or disrupt a gene set in a
host organism in which an increased production of 6-ACA, adipate and/or
HMDA is desired. It is understood that any of the disclosed gene deletion
sets can be used to generate knockout strains with disrupted or deleted
genes that confer increased production of 6-ACA, adipate and/or HMDA.